CD33-Binding Polypeptides and Uses Thereof

ABSTRACT

Provided herein are VHH-containing polypeptides that bind CD33. Uses of the VHH-containing polypeptides are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalApplication No. 62/843,408, filed May 4, 2019, and U.S. ProvisionalApplication No. 62/844,359, filed May 7, 2019, each of which isincorporated by reference herein in its entirety for any purpose.

FIELD

The present invention relates to CD33-binding polypeptides, and methodsof using CD33-binding polypeptides to modulate the biological activityof CD33. Such methods include, but are not limited to, methods oftreating cancer.

BACKGROUND

CD33, also known as p67, SIGLEC3, or SIGLEC-3, binds sialic acids andregulates inflammatory and immune responses of hematopoietic cells. CD33also can influence initiation, proliferation and progression in avariety of hematologic cancers. For example, over 85% of acute myeloidleukemia (AML) cases express the CD33 antigen. Thus, CD33 is a suitabletumor-associated antigen for targeted therapies for hematologic cancers,such as leukemia. Therefore, there exists a therapeutic need for morepotent antagonists of CD33.

SUMMARY

Provided herein are CD33-binding polypeptides and methods of usingCD33-binding polypeptides to treat, for example, leukemia. In someembodiments, a CD33-binding polypeptide comprises at least one VHHdomain. Some embodiments are provided below.

Embodiment 1

A polypeptide comprising at least one VHH domain that binds CD33 andthat comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:47, 3, 7, 11, 15, 19, 23, 27, 31, 35, 50, 53, 56, 59, 62, 65, 68, or 71;a CDR2 comprising the amino acid sequence of SEQ ID NO: 48, 4, 8, 12,16, 20, 24, 28, 32, 36, 51, 54, 57, 60, 63, 66, 69, or 72; and a CDR3comprising the amino acid sequence of SEQ ID NO: 49, 5, 9, 13, 17, 21,25, 29, 33, 37, 52, 55, 58, 61, 64, 67, 70, or 73.

Embodiment 2

The polypeptide of embodiment 1, wherein at least one VHH domaincomprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 47 or3; a CDR2 comprising the amino acid sequence of SEQ ID NO: 48 or 4; anda CDR3 comprising the amino acid sequence of SEQ ID NO: 49 or 5.

Embodiment 3

The polypeptide of embodiment 1 or embodiment 2, wherein at least oneVHH domain comprises a CDR1 comprising the amino acid sequence of SEQ IDNO: 7; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8; and aCDR3 comprising the amino acid sequence of SEQ ID NO: 9.

Embodiment 4

The polypeptide of any one of embodiments 1-3, wherein at least one VHHdomain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:11 or 50; a CDR2 comprising the amino acid sequence of SEQ ID NO: 12 or51; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 13 or52.

Embodiment 5

The polypeptide of any one of embodiments 1-4, wherein at least one VHHdomain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:15, 53, or 56; a CDR2 comprising the amino acid sequence of SEQ ID NO:16, 54, or 57; and a CDR3 comprising the amino acid sequence of SEQ IDNO: 17, 55, or 58.

Embodiment 6

The polypeptide of any one of embodiments 1-5, wherein at least one VHHdomain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:19 or 59; a CDR2 comprising the amino acid sequence of SEQ ID NO: 20 or60; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 21 or61.

Embodiment 7

The polypeptide of any one of embodiments 1-6, wherein at least one VHHdomain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:23 or 62; a CDR2 comprising the amino acid sequence of SEQ ID NO: 24 or63; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25 or64.

Embodiment 8

The polypeptide of any one of embodiments 1-7, wherein at least one VHHdomain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:27 or 65; a CDR2 comprising the amino acid sequence of SEQ ID NO: 28 or66; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 29 or67.

Embodiment 9

The polypeptide of any one of embodiments 1-8, wherein at least one VHHdomain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:31 or 68; a CDR2 comprising the amino acid sequence of SEQ ID NO: 32 or69; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 33 or70.

Embodiment 10

The polypeptide of any one of embodiments 1-9, wherein at least one VHHdomain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:35 or 71; a CDR2 comprising the amino acid sequence of SEQ ID NO: 36 or72; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 37 or73.

Embodiment 11

The polypeptide of any one of embodiments 1-10, wherein at least one VHHdomain comprises a CDR1, a CDR2, and a CDR3, respectively comprising theamino acid sequences of SEQ ID NOs: 47, 48, and 49; SEQ ID NOs: 3, 4,and 5; SEQ ID NOs: 7, 8, and 9; SEQ ID NOs: 11, 12, and 13; SEQ ID NOs:15, 16, and 17; SEQ ID NOs: 19, 20, and 21; SEQ ID NOs: 23, 24, and 25;SEQ ID NOs: 27, 28, and 29; SEQ ID NOs: 31, 32, and 33; SEQ ID NOs: 35,36, and 37; SEQ ID NOs: 50, 51, and 52; SEQ ID NOs: 53, 54, and 55; SEQID NOs: 56, 57, and 58; SEQ ID NOs: 59, 60, and 61; SEQ ID NOs: 62, 63,and 64; SEQ ID NOs: 65, 66, and 67; SEQ ID NOs: 68, 69, and 70; or SEQID NOs: 71, 72, and 73.

Embodiment 12

The polypeptide of any one of embodiments 1-11, wherein at least one VHHdomain is humanized.

Embodiment 13

The polypeptide of any one of embodiments 1-12, wherein at least one VHHdomain comprises an amino acid sequence at least 85%, at least 90%, atleast 95%, or at least 99% identical to the amino acid sequence of SEQID NO: 38, 114, 39, 40, 41, 42, 43, 44, 45, 46, 115, 116, 117, 118, 119,120, 121, or 122.

Embodiment 14

The polypeptide of any one of embodiments 1-12, wherein at least one VHHdomain comprises the amino acid sequence of SEQ ID NO: 38, 114, 39, 40,41, 42, 43, 44, 45, 46, 115, 116, 117, 118, 119, 120, 121, or 122.

Embodiment 15

The polypeptide of any one of embodiment 1-12, wherein at least one VHHdomain comprises an amino acid sequence at least 85%, at least 90%, atleast 95%, or at least 99% identical to the amino acid sequence of SEQID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 123, 124, 125, 126, 127, 128,129, 130, or 131.

Embodiment 16

The polypeptide of any one of embodiments 1-12, wherein at least one VHHdomain comprises the amino acid sequence of SEQ ID NO: 2, 6, 10, 14, 18,22, 26, 30, 34, 123, 124, 125, 126, 127, 128, 129, 130, or 131.

Embodiment 17

The polypeptide of any one of embodiments 1-16, comprising two VHHdomains.

Embodiment 18

The polypeptide of any one of embodiments 1-16, comprising three VHHdomains.

Embodiment 19

The polypeptide of any one of embodiments 1-18, wherein the polypeptidecomprises at least one binding domain that binds an antigen other thanCD33.

Embodiment 20

The polypeptide of embodiment 19, wherein the polypeptide comprises atleast one binding domain that binds CD3, T-cell receptor (TCR) α, TCRβ,CD28, CD16, CD32A, CD64, CD89, NKp46, or NKG2D.

Embodiment 21

The polypeptide of embodiment 17 or 18, wherein each VHH domain bindsCD33.

Embodiment 22

The polypeptide of embodiment 21, wherein each VHH domain comprises thesame CDR1, CDR2, and CDR3 amino acid sequences.

Embodiment 23

The polypeptide of embodiment 21, wherein each VHH domain comprises thesame VHH sequence.

Embodiment 24

The polypeptide of any one of embodiments 1-16, comprising one VHHdomain.

Embodiment 25

The polypeptide of any one of embodiments 1-24, wherein the polypeptidecomprises an Fc region.

Embodiment 26

The polypeptide of embodiment 25, wherein the Fc region comprises anamino acid sequence selected from SEQ ID NOs: 74-109.

Embodiment 27

The polypeptide of embodiment 25 or embodiment 26, which forms a dimerunder physiological conditions.

Embodiment 28

The polypeptide of any one of embodiments 1-27, wherein the CD33 ishuman CD33.

Embodiment 29

The polypeptide of embodiment 28, wherein the human CD33 comprises thesequence of SEQ ID NO: 1.

Embodiment 30

An immunoconjugate comprising the polypeptide of any one of embodiments1-29 and a cytotoxic agent.

Embodiment 31

The immunoconjugate of embodiment 30, wherein the cytotoxic agent isselected from a calicheamicin, an auristatin, a dolastatin, a tubulicin,a maytansinoid, a cryptophycin, a duocarmycin, an esperamicin, apyrrolobenzodiazepine, and an enediyne antibiotic.

Embodiment 32

A pharmaceutical composition comprising the polypeptide of any one ofembodiments 1-29 or the immunoconjugate of embodiment 30 or embodiment31, and a pharmaceutically acceptable carrier.

Embodiment 33

An isolated nucleic acid that encodes the polypeptide of any one ofembodiments 1-29.

Embodiment 34

A vector comprising the nucleic acid of embodiment 33.

Embodiment 35

A host cell comprising the nucleic acid of embodiment 33 or the vectorof embodiment 34.

Embodiment 36

A host cell that expresses the polypeptide of any one of embodiments1-29.

Embodiment 37

A method of producing the polypeptide of any one of embodiments 1-29,comprising incubating the host cell of embodiment 35 or embodiment 36under conditions suitable for expression of the polypeptide.

Embodiment 38

The method of embodiment 37, further comprising isolating thepolypeptide.

Embodiment 39

A method of treating cancer comprising administering to a subject withcancer a pharmaceutically effective amount of the polypeptide of any oneof embodiments 1-29, the immunoconjugate of embodiment 30 or embodiment31, or the pharmaceutical composition of embodiment 32.

Embodiment 40

The method of embodiment 39, wherein the cancer is selected fromlymphoma; Hodgkin's lymphoma; non-Hodgkin's lymphoma; B-cell lymphoma;low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic(SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuseNHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; highgrade small non-cleaved cell NHL; bulky disease NHL; mantle celllymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia;chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL);acute myeloid leukemia (AML); Hairy cell leukemia; and chronicmyeloblastic leukemia.

Embodiment 41

The method of embodiment 39 or 40, wherein the cancer is acute myeloidleukemia (AML).

Embodiment 42

The method of any one of embodiments 39-41, further comprisingadministering an additional therapeutic agent.

Embodiment 43

The method of embodiment 42, wherein the additional therapeutic agent isan anti-cancer agent.

Embodiment 44

The method of embodiment 43, wherein the anti-cancer agent is selectedfrom a chemotherapeutic agent, an anti-cancer biologic, radiationtherapy, CAR-T therapy, and an oncolytic virus.

Embodiment 45

The method of any one of embodiments 39-44, wherein the cancer is aCD33-expressing cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows Bio-Layer Interferometry data for hz1E4v2 compared to otherCD33-binding sdAbs described herein.

FIG. 2A-2M show binding of certain single-domain antibodies (sdAbs) toCD33 expressed on MOLM-13 or HEK 293 cells. “MOLM-13” indicates MOL-13cells that have been transfected with a plasmid encoding CD33, asdescribed in Example 2. “CD33m” indicates a truncated CD33 protein (SEQID NO: 113). “CD33M” indicates a full length CD33 protein (SEQ ID NO:112). “HEK 293” or “Parental HEK 293” indicates untransfected HEK 293cells. FIG. 2A shows binding of 1E4-IgG1 to CD33. FIG. 2B shows bindingof hz1E4v2-IgG1 to CD33. FIG. 2C shows binding of 1H9-IgG1 to CD33. FIG.2D shows binding of hz1H9v2-IgG1 to CD33. FIG. 2E shows binding ofhz1G3v3-IgG1 to CD33. FIG. 2F shows binding of 1C7-IgG1 to CD33. FIG. 2Gshows binding of hz1C7v1-IgG1 to CD33. FIG. 2H shows binding ofhz1C7v11-IgG1 to CD33. FIG. 2I shows binding of hzA07v4-IgG1 to CD33.FIG. 2J shows binding of hzB07v7-IgG1 to CD33. FIG. 2K shows binding ofhzG11v2-IgG1 to CD33. FIG. 2L shows binding of F02-IgG1 to CD33. FIG. 2Mshows binding of hzF02v18-IgG1 to CD33.

DETAILED DESCRIPTION

Embodiments provided herein relate to CD33-binding polypeptides andtheir use in various methods of treating cancer.

Definitions and Various Embodiments

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

All references cited herein, including patent applications, patentpublications, and Genbank Accession numbers are herein incorporated byreference, as if each individual reference were specifically andindividually indicated to be incorporated by reference in its entirety.

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3rd. edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS INMOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (2003)); the seriesMETHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICALAPPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMALCELL CULTURE (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; CellBiology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press;Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Celland Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;Cell and Tissue Culture Laboratory Procedures (A. Doyle, J. B.Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbookof Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos,eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds.,1994); Current Protocols in Immunology (J. E. Coligan et al., eds.,1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRLPress, 1988-1989); Monoclonal Antibodies: A Practical Approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); UsingAntibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold SpringHarbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D.Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principlesand Practice of Oncology (V. T. DeVita et al., eds., J.B. LippincottCompany, 1993); and updated versions thereof.

Unless otherwise defined, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context or expressly indicated, singularterms shall include pluralities and plural terms shall include thesingular. For any conflict in definitions between various sources orreferences, the definition provided herein will control.

In general, the numbering of the residues in an immunoglobulin heavychain is that of the EU index as in Kabat et al., Sequences of Proteinsof Immunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991). The “EU index as in Kabat”refers to the residue numbering of the human IgG1 EU antibody.

It is understood that embodiments of the invention described hereininclude “consisting” and/or “consisting essentially of” embodiments. Asused herein, the singular form “a”, “an”, and “the” includes pluralreferences unless indicated otherwise. Use of the term “or” herein isnot meant to imply that alternatives are mutually exclusive.

In this application, the use of “or” means “and/or” unless expresslystated or understood by one skilled in the art. In the context of amultiple dependent claim, the use of “or” refers back to more than onepreceding independent or dependent claim.

The phrase “reference sample”, “reference cell”, or “reference tissue”,denote a sample with at least one known characteristic that can be usedas a comparison to a sample with at least one unknown characteristic. Insome embodiments, a reference sample can be used as a positive ornegative indicator. A reference sample can be used to establish a levelof protein and/or mRNA that is present in, for example, healthy tissue,in contrast to a level of protein and/or mRNA present in the sample withunknown characteristics. In some embodiments, the reference sample comesfrom the same subject, but is from a different part of the subject thanthat being tested. In some embodiments, the reference sample is from atissue area surrounding or adjacent to the cancer. In some embodiments,the reference sample is not from the subject being tested, but is asample from a subject known to have, or not to have, a disorder inquestion (for example, a particular cancer or CD33-related disorder). Insome embodiments, the reference sample is from the same subject, butfrom a point in time before the subject developed cancer. In someembodiments, the reference sample is from a benign cancer sample, fromthe same or a different subject. When a negative reference sample isused for comparison, the level of expression or amount of the moleculein question in the negative reference sample will indicate a level atwhich one of skill in the art will appreciate, given the presentdisclosure, that there is no and/or a low level of the molecule. When apositive reference sample is used for comparison, the level ofexpression or amount of the molecule in question in the positivereference sample will indicate a level at which one of skill in the artwill appreciate, given the present disclosure, that there is a level ofthe molecule.

The terms “benefit”, “clinical benefit”, “responsiveness”, and“therapeutic responsiveness” as used herein in the context of benefitingfrom or responding to administration of a therapeutic agent, can bemeasured by assessing various endpoints, e.g., inhibition, to someextent, of disease progression, including slowing down and completearrest; reduction in the number of disease episodes and/or symptoms;reduction in lesion size; inhibition (that is, reduction, slowing downor complete stopping) of disease cell infiltration into adjacentperipheral organs and/or tissues; inhibition (that is, reduction,slowing down or complete stopping) of disease spread; relief, to someextent, of one or more symptoms associated with the disorder; increasein the length of disease-free presentation following treatment, forexample, progression-free survival; increased overall survival; higherresponse rate; and/or decreased mortality at a given point of timefollowing treatment. A subject or cancer that is “non-responsive” or“fails to respond” is one that has failed to meet the above notedqualifications to be “responsive”.

The terms “nucleic acid molecule”, “nucleic acid” and “polynucleotide”may be used interchangeably, and refer to a polymer of nucleotides. Suchpolymers of nucleotides may contain natural and/or non-naturalnucleotides, and include, but are not limited to, DNA, RNA, and PNA.“Nucleic acid sequence” refers to the linear sequence of nucleotidescomprised in the nucleic acid molecule or polynucleotide.

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues, and are not limited to a minimumlength. Such polymers of amino acid residues may contain natural ornon-natural amino acid residues, and include, but are not limited to,peptides, oligopeptides, dimers, trimers, and multimers of amino acidresidues. Both full-length proteins and fragments thereof areencompassed by the definition. The terms also include post-expressionmodifications of the polypeptide, for example, glycosylation,sialylation, acetylation, phosphorylation, and the like. Furthermore,for purposes of the present disclosure, a “polypeptide” refers to aprotein which includes modifications, such as deletions, additions, andsubstitutions (generally conservative in nature), to the nativesequence, as long as the protein maintains the desired activity. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of hosts which producethe proteins or errors due to PCR amplification.

“CD33” as used herein refers to any native, mature CD33 that resultsfrom processing of a CD33 precursor in a cell. The term includes CD33from any vertebrate source, including mammals such as primates (e.g.,humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice andrats), unless otherwise indicated. The term also includesnaturally-occurring variants of CD33, such as splice variants or allelicvariants. A nonlimiting exemplary human CD33 amino acid sequence isshown, e.g., in UniProt Accession No. P20138. A mature form of CD33 maylack a signal peptide (for example, a mature form of CD33 may lack aminoacids 1-17 of UniProt Accession No. P20138). See SEQ ID NO. 1.

The term “specifically binds” to an antigen or epitope is a term that iswell understood in the art, and methods to determine such specificbinding are also well known in the art. A molecule is said to exhibit“specific binding” or “preferential binding” if it reacts or associatesmore frequently, more rapidly, with greater duration and/or with greateraffinity with a particular cell or substance than it does withalternative cells or substances. A single-domain antibody (sdAb) orVHH-containing polypeptide “specifically binds” or “preferentiallybinds” to a target if it binds with greater affinity, avidity, morereadily, and/or with greater duration than it binds to other substances.For example, a sdAb or VHH-containing polypeptide that specifically orpreferentially binds to a CD33 epitope is a sdAb or VHH-containingpolypeptide that binds this epitope with greater affinity, avidity, morereadily, and/or with greater duration than it binds to other CD33epitopes or non-CD33 epitopes. It is also understood by reading thisdefinition that; for example, a sdAb or VHH-containing polypeptide thatspecifically or preferentially binds to a first target may or may notspecifically or preferentially bind to a second target. As such,“specific binding” or “preferential binding” does not necessarilyrequire (although it can include) exclusive binding. Generally, but notnecessarily, reference to binding means preferential binding.“Specificity” refers to the ability of a binding protein to selectivelybind an antigen.

The terms “inhibition” or “inhibit” refer to a decrease or cessation ofany phenotypic characteristic or to the decrease or cessation in theincidence, degree, or likelihood of that characteristic. To “reduce” or“inhibit” is to decrease, reduce or arrest an activity, function, and/oramount as compared to a reference. In some embodiments, by “reduce” or“inhibit” is meant the ability to cause an overall decrease of 10% orgreater. In some embodiments, by “reduce” or “inhibit” is meant theability to cause an overall decrease of 50% or greater. In someembodiments, by “reduce” or “inhibit” is meant the ability to cause anoverall decrease of 75%, 85%, 90%, 95%, or greater. In some embodiments,the amount noted above is inhibited or decreased over a period of time,relative to a control over the same period of time. As used herein, theterm “inhibit” with regard to the activity of CD33 refers to a decreasein an activity of CD33, such as binding to a sialic acid. In someembodiments, “inhibit” refers to a decrease in a CD33 activity comparedto the CD33 activity in the absence of the modulator. In someembodiments, a CD33-binding polypeptide described herein inhibitsbinding of CD33 to a sialic acid.

As used herein, the term “epitope” refers to a site on a target molecule(for example, an antigen, such as a protein, nucleic acid, carbohydrateor lipid) to which an antigen-binding molecule (for example, a sdAb orVHH-containing polypeptide) binds. Epitopes often include a chemicallyactive surface grouping of molecules such as amino acids, polypeptidesor sugar side chains and have specific three-dimensional structuralcharacteristics as well as specific charge characteristics. Epitopes canbe formed both from contiguous and/or juxtaposed noncontiguous residues(for example, amino acids, nucleotides, sugars, lipid moiety) of thetarget molecule. Epitopes formed from contiguous residues (for example,amino acids, nucleotides, sugars, lipid moiety) typically are retainedon exposure to denaturing solvents whereas epitopes formed by tertiaryfolding typically are lost on treatment with denaturing solvents. Anepitope may include but is not limited to at least 3, at least 5 or 8-10residues (for example, amino acids or nucleotides). In some embodiments,an epitope is less than 20 residues (for example, amino acids ornucleotides) in length, less than 15 residues or less than 12 residues.Two antibodies may bind the same epitope within an antigen if theyexhibit competitive binding for the antigen. In some embodiments, anepitope can be identified by a certain minimal distance to a CDR residueon the antigen-binding molecule. In some embodiments, an epitope can beidentified by the above distance, and further limited to those residuesinvolved in a bond (for example, a hydrogen bond) between a residue ofthe antigen-binding molecule and an antigen residue. An epitope can beidentified by various scans as well, for example an alanine or argininescan can indicate one or more residues that the antigen-binding moleculecan interact with. Unless explicitly denoted, a set of residues as anepitope does not exclude other residues from being part of the epitopefor a particular antigen-binding molecule. Rather, the presence of sucha set designates a minimal series (or set of species) of epitopes. Thus,in some embodiments, a set of residues identified as an epitopedesignates a minimal epitope of relevance for the antigen, rather thanan exclusive list of residues for an epitope on an antigen.

A “nonlinear epitope” or “conformational epitope” comprisesnoncontiguous polypeptides, amino acids and/or sugars within theantigenic protein to which an antigen-binding molecule specific to theepitope binds. In some embodiments, at least one of the residues will benoncontiguous with the other noted residues of the epitope; however, oneor more of the residues can also be contiguous with the other residues.

A “linear epitope” comprises contiguous polypeptides, amino acids and/orsugars within the antigenic protein to which an antigen-binding moleculespecific to the epitope binds. It is noted that, in some embodiments,not every one of the residues within the linear epitope need be directlybound (or involved in a bond) by the antigen-binding molecule. In someembodiments, linear epitopes can be from immunizations with a peptidethat effectively consisted of the sequence of the linear epitope, orfrom structural sections of a protein that are relatively isolated fromthe remainder of the protein (such that the antigen-binding molecule caninteract, at least primarily), just with that sequence section.

The term “antibody” is used in the broadest sense and encompass variouspolypeptides that comprise antibody-like antigen-binding domains,including but not limited to conventional antibodies (typicallycomprising at least one heavy chain and at least one light chain),single-domain antibodies (sdAbs, comprising at least one VHH domain andan Fc region), VHH-containing polypeptides (polypeptides comprising atleast one VHH domain), and fragments of any of the foregoing so long asthey exhibit the desired antigen-binding activity. In some embodiments,an antibody comprises a dimerization domain. Such dimerization domainsinclude, but are not limited to, heavy chain constant domains(comprising CH1, hinge, CH2, and CH3, where CH1 typically pairs with alight chain constant domain, CL, while the hinge mediates dimerization)and Fc regions (comprising hinge, CH2, and CH3, where the hinge mediatesdimerization).

The term antibody also includes, but is not limited to, chimericantibodies, humanized antibodies, and antibodies of various species suchas camelid (including llama), shark, mouse, human, cynomolgus monkey,etc.

The term “antigen-binding domain” as used herein refers to a portion ofan antibody sufficient to bind antigen. In some embodiments, an antigenbinding domain of a conventional antibody comprises three heavy chainCDRs and three light chain CDRs. Thus, in some embodiments, an antigenbinding domain comprises a heavy chain variable region comprisingCDR1-FR2-CDR2-FR3-CDR3, and any portions of FR1 and/or FR4 required tomaintain binding to antigen, and a light chain variable regioncomprising CDR1-FR2-CDR2-FR3-CDR3, and any portions of FR1 and/or FR4required to maintain binding to antigen. In some embodiments, anantigen-binding domain of an sdAb or VHH-containing polypeptidecomprises three CDRs of a VHH domain. Thus, in some embodiments, anantigen binding domain of an sdAb or VHH-containing polypeptidecomprises a VHH domain comprising CDR1-FR2-CDR2-FR3-CDR3, and anyportions of FR1 and/or FR4 required to maintain binding to antigen.

The term “VHH” or “VHH domain” or “VHH antigen-binding domain” as usedherein refers to the antigen-binding portion of a single-domainantibody, such as a camelid antibody or shark antibody. In someembodiments, a VHH comprises three CDRs and four framework regions,designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In someembodiments, a VHH may be truncated at the N-terminus or C-terminus suchthat it comprises only a partial FR1 and/or FR4, or lacks one or both ofthose framework regions, so long as the VHH substantially maintainsantigen binding and specificity.

The terms “single domain antibody” and “sdAb” are used interchangeablyherein to refer to an antibody comprising at least one monomeric domain,such as a VHH domain, without a light chain, and an Fc region. In someembodiments, an sdAb is a dimer of two polypeptides wherein eachpolypeptide comprises at least one VHH domain and an Fc region. As usedherein, the terms “single domain antibody” and “sdAb” encompasspolypeptides that comprise multiple VHH domains, such as a polypeptidehaving the structure VHH₁-VHH₂-Fc or VHH₁-VHH₂-VHH₃-Fc, wherein VHH₁,VHH₂, and VHH₃ may be the same or different.

The term “VHH-containing polypeptide” refers to a polypeptide thatcomprises at least one VHH domain. In some embodiments, a VHHpolypeptide comprises two, three, or four or more VHH domains, whereineach VHH domain may be the same or different. In some embodiments, aVHH-containing polypeptide comprises an Fc region. In some suchembodiments, the VHH-containing polypeptide may be referred to as ansdAb. Further, in some such embodiments, the VHH polypeptide may form adimer. Nonlimiting structures of VHH-containing polypeptides, which arealso sdAbs, include VHH₁-Fc, VHH₁-VHH₂-Fc, and VHH₁-VHH₂-VHH₃-Fc,wherein VHH₁, VHH₂, and VHH₃ may be the same or different. In someembodiments of such structures, one VHH may be connected to another VHHby a linker, or one VHH may be connected to the Fc by a linker. In somesuch embodiments, the linker comprises 1-20 amino acids, preferably 1-20amino acids predominantly composed of glycine and, optionally, serine.In some embodiments, when a VHH-containing polypeptide comprises an Fc,it forms a dimer. Thus, the structure VHH₁-VHH₂-Fc, if it forms a dimer,is considered to be tetravalent (i.e., the dimer has four VHH domains).Similarly, the structure VHH₁-VHH₂-VHH₃-Fc, if it forms a dimer, isconsidered to be hexavalent (i.e., the dimer has six VHH domains).

The term “monoclonal antibody” refers to an antibody (including an sdAbor VHH-containing polypeptide) of a substantially homogeneous populationof antibodies, that is, the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts. Monoclonal antibodiesare highly specific, being directed against a single antigenic site.Furthermore, in contrast to polyclonal antibody preparations, whichtypically include different antibodies directed against differentdeterminants (epitopes), each monoclonal antibody is directed against asingle determinant on the antigen. Thus, a sample of monoclonalantibodies can bind to the same epitope on the antigen. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies may be made by thehybridoma method first described by Kohler and Milstein, 1975, Nature256:495, or may be made by recombinant DNA methods such as described inU.S. Pat. No. 4,816,567. The monoclonal antibodies may also be isolatedfrom phage libraries generated using the techniques described inMcCafferty et al., 1990, Nature 348:552-554, for example.

The term “CDR” denotes a complementarity determining region as definedby at least one manner of identification to one of skill in the art. Insome embodiments, CDRs can be defined in accordance with any of theChothia numbering schemes, the Kabat numbering scheme, a combination ofKabat and Chothia, the AbM definition, and/or the contact definition. AVHH comprises three CDRs, designated CDR1, CDR2, and CDR3.

The term “heavy chain constant region” as used herein refers to a regioncomprising at least three heavy chain constant domains, CH1, hinge, CH2,and CH3. Of course, non-function-altering deletions and alterationswithin the domains are encompassed within the scope of the term “heavychain constant region,” unless designated otherwise. Nonlimitingexemplary heavy chain constant regions include γ, δ, and α. Nonlimitingexemplary heavy chain constant regions also include ε and μ. Each heavyconstant region corresponds to an antibody isotype. For example, anantibody comprising a γ constant region is an IgG antibody, an antibodycomprising a δ constant region is an IgD antibody, and an antibodycomprising an α constant region is an IgA antibody. Further, an antibodycomprising a μ constant region is an IgM antibody, and an antibodycomprising an ε constant region is an IgE antibody. Certain isotypes canbe further subdivided into subclasses. For example, IgG antibodiesinclude, but are not limited to, IgG1 (comprising a γ₁ constant region),IgG2 (comprising a γ₂ constant region), IgG3 (comprising a γ₃ constantregion), and IgG4 (comprising a γ₄ constant region) antibodies; IgAantibodies include, but are not limited to, IgA1 (comprising an α₁constant region) and IgA2 (comprising an α₂ constant region) antibodies;and IgM antibodies include, but are not limited to, IgM1 and IgM2.

A “Fc region” as used herein refers to a portion of a heavy chainconstant region comprising CH2 and CH3. In some embodiments, an Fcregion comprises a hinge, CH2, and CH3. In various embodiments, when anFc region comprises a hinge, the hinge mediates dimerization between twoFc-containing polypeptides. An Fc region may be of any antibody heavychain constant region isotype discussed herein. In some embodiments, anFc region is an IgG1, IgG2, IgG3, or IgG4.

An “acceptor human framework” as used herein is a framework comprisingthe amino acid sequence of a heavy chain variable domain (V_(H))framework derived from a human immunoglobulin framework or a humanconsensus framework, as discussed herein. An acceptor human frameworkderived from a human immunoglobulin framework or a human consensusframework can comprise the same amino acid sequence thereof, or it cancontain amino acid sequence changes. In some embodiments, the number ofamino acid changes are fewer than 10, or fewer than 9, or fewer than 8,or fewer than 7, or fewer than 6, or fewer than 5, or fewer than 4, orfewer than 3, across all of the human frameworks in a single antigenbinding domain, such as a VHH.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (for example,an antibody, such as an sdAb, or VHH-containing polypeptide) and itsbinding partner (for example, an antigen). The affinity or the apparentaffinity of a molecule X for its partner Y can generally be representedby the dissociation constant (K_(d)) or the K_(d-apparent),respectively. Affinity can be measured by common methods known in theart (such as, for example, ELISA K_(d), KinExA, flow cytometry, and/orsurface plasmon resonance devices), including those described herein.Such methods include, but are not limited to, methods involvingBIAcore®, Octet®, or flow cytometry.

The term “K_(d)”, as used herein, refers to the equilibrium dissociationconstant of an antigen-binding molecule/antigen interaction. When theterm “K_(d)” is used herein, it includes K_(d) and K_(d-apparent).

In some embodiments, the K_(d) of the antigen-binding molecule ismeasured by flow cytometry using an antigen-expressing cell line andfitting the mean fluorescence measured at each antibody concentration toa non-linear one-site binding equation (Prism Software graphpad). Insome such embodiments, the K_(d) is K_(d)-apparent.

The term “biological activity” refers to any one or more biologicalproperties of a molecule (whether present naturally as found in vivo, orprovided or enabled by recombinant means).

An “agonist” or “activating” antibody is one that increases and/oractivates a biological activity of the target antigen. In someembodiments, the agonist antibody binds to an antigen and increases itsbiologically activity by at least about 20%, 40%, 60%, 80%, 85% or more.

An “antagonist”, a “blocking” or “neutralizing” antibody is one thatinhibits, decreases and/or inactivates a biological activity of thetarget antigen. In some embodiments, the neutralizing antibody binds toan antigen and reduces its biologically activity by at least about 20%,40%, 60%, 80%, 85% 90%, 95%, 99% or more.

An “affinity matured” sdAb or VHH-containing polypeptide refers to asdAb or VHH-containing polypeptide with one or more alterations in oneor more CDRs compared to a parent sdAb or VHH-containing polypeptidethat does not possess such alterations, such alterations resulting in animprovement in the affinity of the sdAb or VHH-containing polypeptidefor antigen.

A “humanized VHH” as used herein refers to a VHH in which one or moreframework regions have been substantially replaced with human frameworkregions. In some instances, certain framework region (FR) residues ofthe human immunoglobulin are replaced by corresponding non-humanresidues. Furthermore, the humanized VHH can comprise residues that arefound neither in the original VHH nor in the human framework sequences,but are included to further refine and optimize sdAb VHH-containingpolypeptide performance. In some embodiments, a humanized sdAb orVHH-containing polypeptide comprises a human Fc region. As will beappreciated, a humanized sequence can be identified by its primarysequence and does not necessarily denote the process by which theantibody was created.

An “effector-positive Fc region” possesses an “effector function” of anative sequence Fc region. Exemplary “effector functions” include Fcreceptor binding; C1q binding and complement dependent cytotoxicity(CDC); Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; down regulation of cell surfacereceptors (for example B-cell receptor); and B-cell activation, etc.Such effector functions generally require the Fc region to be combinedwith a binding domain (for example, an antibody variable domain) and canbe assessed using various assays.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include a native sequence human IgG1 Fc region(non-A and A allotypes); native sequence human IgG2 Fc region; nativesequence human IgG3 Fc region; and native sequence human IgG4 Fc regionas well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification. In some embodiments, a “variant Fc region” comprisesan amino acid sequence which differs from that of a native sequence Fcregion by virtue of at least one amino acid modification, yet retains atleast one effector function of the native sequence Fc region. In someembodiments, the variant Fc region has at least one amino acidsubstitution compared to a native sequence Fc region or to the Fc regionof a parent polypeptide, for example, from about one to about ten aminoacid substitutions, and preferably, from about one to about five aminoacid substitutions in a native sequence Fc region or in the Fc region ofthe parent polypeptide. In some embodiments, the variant Fc regionherein will possess at least about 80% sequence identity with a nativesequence Fc region and/or with an Fc region of a parent polypeptide, atleast about 90% sequence identity therewith, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% sequence identity therewith.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. In some embodiments, an FcγR is a native human FcR. Insome embodiments, an FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof those receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domainInhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (See, for example,Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, forexample, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capelet al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin.Med. 126:330-41 (1995). Other FcRs, including those to be identified inthe future, are encompassed by the term “FcR” herein. For example, theterm “Fc receptor” or “FcR” also includes the neonatal receptor, FcRn,which is responsible for the transfer of maternal IgGs to the fetus(Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.24:249 (1994)) and regulation of homeostasis of immunoglobulins. Methodsof measuring binding to FcRn are known (see, for example, Ghetie andWard, Immunol. Today 18(12):592-598 (1997); Ghetie et al., NatureBiotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem.279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al.).

A “chimeric antigen receptor” as used herein refers to an engineeredpolypeptide that comprises an extracellular antigen recognition domain,a transmembrane domain, and an intracellular signaling domain. In someembodiments, the extracellular antigen recognition domain comprises aVHH domain.

The term “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between two ormore numeric values such that one of skill in the art would consider thedifference between the two or more values to be of little or nobiological and/or statistical significance within the context of thebiological characteristic measured by said value. In some embodimentsthe two or more substantially similar values differ by no more thanabout any one of 5%, 10%, 15%, 20%, 25%, or 50%.

A polypeptide “variant” means a biologically active polypeptide havingat least about 80% amino acid sequence identity with the native sequencepolypeptide after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Such variants include, for instance, polypeptides wherein oneor more amino acid residues are added, or deleted, at the N- orC-terminus of the polypeptide. In some embodiments, a variant will haveat least about 80% amino acid sequence identity. In some embodiments, avariant will have at least about 90% amino acid sequence identity. Insome embodiments, a variant will have at least about 95% amino acidsequence identity with the native sequence polypeptide.

As used herein, “percent (%) amino acid sequence identity” and“homology” with respect to a peptide, polypeptide or antibody sequenceare defined as the percentage of amino acid residues in a candidatesequence that are identical with the amino acid residues in the specificpeptide or polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.

An amino acid substitution may include but are not limited to thereplacement of one amino acid in a polypeptide with another amino acid.Exemplary substitutions are shown in Table 1. Amino acid substitutionsmay be introduced into an antibody of interest and the products screenedfor a desired activity, for example, retained/improved antigen binding,decreased immunogenicity, or improved ADCC or CDC.

TABLE 1 Original Residue Exemplary Substitutions Ala (A) Val; Leu; IleArg (R) Lys; Gln; Asn Asn (N) Gln; His; Asp, Lys; Arg Asp (D) Glu; AsnCys (C) Ser; Ala Gln (Q) Asn; Glu Glu (E) Asp; Gln Gly (G) Ala His (H)Asn; Gln; Lys; Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu (L)Norleucine; Ile; Val; Met; Ala; Phe Lys (K) Arg; Gln; Asn Met (M) Leu;Phe; Ile Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Pro (P) Ala Ser (S) ThrThr (T) Val; Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe; Thr; Ser Val (V)Ile; Leu; Met; Phe; Ala; Norleucine

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

The term “vector” is used to describe a polynucleotide that can beengineered to contain a cloned polynucleotide or polynucleotides thatcan be propagated in a host cell. A vector can include one or more ofthe following elements: an origin of replication, one or more regulatorysequences (such as, for example, promoters and/or enhancers) thatregulate the expression of the polypeptide of interest, and/or one ormore selectable marker genes (such as, for example, antibioticresistance genes and genes that can be used in colorimetric assays, forexample, β-galactosidase). The term “expression vector” refers to avector that is used to express a polypeptide of interest in a host cell.

A “host cell” refers to a cell that may be or has been a recipient of avector or isolated polynucleotide. Host cells may be prokaryotic cellsor eukaryotic cells. Exemplary eukaryotic cells include mammalian cells,such as primate or non-primate animal cells; fungal cells, such asyeast; plant cells; and insect cells. Nonlimiting exemplary mammaliancells include, but are not limited to, NSO cells, PER.C6® cells(Crucell), and 293 and CHO cells, and their derivatives, such as 293-6E,CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells. Host cells include progeny ofa single host cell, and the progeny may not necessarily be completelyidentical (in morphology or in genomic DNA complement) to the originalparent cell due to natural, accidental, or deliberate mutation. A hostcell includes cells transfected in vivo with a polynucleotide(s) aprovided herein.

The term “isolated” as used herein refers to a molecule that has beenseparated from at least some of the components with which it istypically found in nature or produced. For example, a polypeptide isreferred to as “isolated” when it is separated from at least some of thecomponents of the cell in which it was produced. Where a polypeptide issecreted by a cell after expression, physically separating thesupernatant containing the polypeptide from the cell that produced it isconsidered to be “isolating” the polypeptide. Similarly, apolynucleotide is referred to as “isolated” when it is not part of thelarger polynucleotide (such as, for example, genomic DNA ormitochondrial DNA, in the case of a DNA polynucleotide) in which it istypically found in nature, or is separated from at least some of thecomponents of the cell in which it was produced, for example, in thecase of an RNA polynucleotide. Thus, a DNA polynucleotide that iscontained in a vector inside a host cell may be referred to as“isolated”.

The terms “individual” and “subject” are used interchangeably herein torefer to an animal; for example, a mammal. In some embodiments, methodsof treating mammals, including, but not limited to, humans, rodents,simians, felines, canines, equines, bovines, porcines, ovines, caprines,mammalian laboratory animals, mammalian farm animals, mammalian sportanimals, and mammalian pets, are provided. In some examples, an“individual” or “subject” refers to an individual or subject in need oftreatment for a disease or disorder. In some embodiments, the subject toreceive the treatment can be a patient, designating the fact that thesubject has been identified as having a disorder of relevance to thetreatment, or being at adequate risk of contracting the disorder.

A “disease” or “disorder” as used herein refers to a condition wheretreatment is needed and/or desired.

The term “tumor cell”, “cancer cell”, “cancer”, “tumor”, and/or“neoplasm”, unless otherwise designated, are used herein interchangeablyand refer to a cell (or cells) exhibiting an uncontrolled growth and/orabnormal increased cell survival and/or inhibition of apoptosis whichinterferes with the normal functioning of bodily organs and systems.Included in this definition are benign and malignant cancers,hematologic cancers such as leukemias, lymphomas, and multiple myelomas,polyps, hyperplasia, as well as dormant tumors or micrometastases.

The terms “cancer” and “tumor” encompass solid andhematological/lymphatic cancers and also encompass malignant,pre-malignant, and benign growth, such as dysplasia. Exemplary cancersinclude, but are not limited to: basal cell carcinoma, biliary tractcancer; bladder cancer; bone cancer; brain and central nervous systemcancer; breast cancer; cancer of the peritoneum; cervical cancer;choriocarcinoma; colon and rectum cancer; connective tissue cancer;cancer of the digestive system; endometrial cancer; esophageal cancer;eye cancer; cancer of the head and neck; gastric cancer (includinggastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma;intra-epithelial neoplasm; kidney or renal cancer; larynx cancer;leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer,non-small cell lung cancer, adenocarcinoma of the lung, and squamouscarcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavitycancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreaticcancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectalcancer; cancer of the respiratory system; salivary gland carcinoma;sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicularcancer; thyroid cancer; uterine or endometrial cancer; cancer of theurinary system; vulval cancer; lymphoma including Hodgkin's andnon-Hodgkin's lymphoma, as well as B-cell lymphoma (including lowgrade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL)NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL;high grade immunoblastic NHL; high grade lymphoblastic NHL; high gradesmall non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; acutemyeloid leukemia (AML); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; as well as other carcinomas and sarcomas; and post-transplantlymphoproliferative disorder (PTLD), as well as abnormal vascularproliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome.

The term “non-tumor cell” or “non-cancer cell” as used herein refers toa normal cells or tissue. Exemplary non-tumor cells include, but are notlimited to: T-cells, B-cells, natural killer (NK) cells, natural killerT (NKT) cells, dendritic cells, monocytes, macrophages, epithelialcells, fibroblasts, hepatocytes, interstitial kidney cells,fibroblast-like synoviocytes, osteoblasts, and cells located in thebreast, skeletal muscle, pancreas, stomach, ovary, small intestines,placenta, uterus, testis, kidney, lung, heart, brain, liver, prostate,colon, lymphoid organs, bone, and bone-derived mesenchymal stem cells.The term “a cell or tissue located in the periphery” as used hereinrefers to non-tumor cells not located near tumor cells and/or within thetumor microenvironment.

The term “cells or tissue within the tumor microenvironment” as usedherein refers to the cells, molecules, extracellular matrix and/or bloodvessels that surround and/or feed a tumor cell. Exemplary cells ortissue within the tumor microenvironment include, but are not limitedto: tumor vasculature; tumor-infiltrating lymphocytes; fibroblastreticular cells; endothelial progenitor cells (EPC); cancer-associatedfibroblasts; pericytes; other stromal cells; components of theextracellular matrix (ECM); dendritic cells; antigen presenting cells;T-cells; regulatory T-cells (Treg cells); macrophages; neutrophils;myeloid-derived suppressor cells (MDSCs) and other immune cells locatedproximal to a tumor. Methods for identifying tumor cells, and/orcells/tissues located within the tumor microenvironment are well knownin the art, as described herein, below.

In some embodiments, an “increase” or “decrease” refers to astatistically significant increase or decrease, respectively. As will beclear to the skilled person, “modulating” can also involve effecting achange (which can either be an increase or a decrease) in affinity,avidity, specificity and/or selectivity of a target or antigen, for oneor more of its ligands, binding partners, partners for association intoa homomultimeric or heteromultimeric form, or substrates; effecting achange (which can either be an increase or a decrease) in thesensitivity of the target or antigen for one or more conditions in themedium or surroundings in which the target or antigen is present (suchas pH, ion strength, the presence of co-factors, etc.); and/or cellularproliferation or cytokine production, compared to the same conditionsbut without the presence of a test agent. This can be determined in anysuitable manner and/or using any suitable assay known per se ordescribed herein, depending on the target involved.

As used herein, “an immune response” is meant to encompass cellularand/or humoral immune responses that are sufficient to inhibit orprevent onset or ameliorate the symptoms of disease (for example, canceror cancer metastasis). “An immune response” can encompass aspects ofboth the innate and adaptive immune systems.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. “Treatment” as used herein, covers anyadministration or application of a therapeutic for disease in a mammal,including a human. For purposes of this disclosure, beneficial ordesired clinical results include, but are not limited to, any one ormore of: alleviation of one or more symptoms, diminishment of extent ofdisease, preventing or delaying spread (for example, metastasis, forexample metastasis to the lung or to the lymph node) of disease,preventing or delaying recurrence of disease, delay or slowing ofdisease progression, amelioration of the disease state, inhibiting thedisease or progression of the disease, inhibiting or slowing the diseaseor its progression, arresting its development, and remission (whetherpartial or total). Also encompassed by “treatment” is a reduction ofpathological consequence of a proliferative disease. The methodsprovided herein contemplate any one or more of these aspects oftreatment. In-line with the above, the term treatment does not requireone-hundred percent removal of all aspects of the disorder.

“Ameliorating” means a lessening or improvement of one or more symptomsas compared to not administering a therapeutic agent. “Ameliorating”also includes shortening or reduction in duration of a symptom.

The term “anti-cancer agent” is used herein in its broadest sense torefer to agents that are used in the treatment of one or more cancers.Exemplary classes of such agents in include, but are not limited to,chemotherapeutic agents, anti-cancer biologics (such as cytokines,receptor extracellular domain-Fc fusions, and antibodies), radiationtherapy, CAR-T therapy, therapeutic oligonucleotides (such as antisenseoligonucleotides and siRNAs) and oncolytic viruses.

The term “biological sample” means a quantity of a substance from aliving thing or formerly living thing. Such substances include, but arenot limited to, blood, (for example, whole blood), plasma, serum, urine,amniotic fluid, synovial fluid, endothelial cells, leukocytes,monocytes, other cells, organs, tissues, bone marrow, lymph nodes andspleen.

The term “control” or “reference” in the context of an experiment orcomparison, refers to a composition known to not contain an analyte(“negative control”) or to contain an analyte (“positive control”). Apositive control can comprise a known concentration of analyte. Acontrol or reference may also refer to a control agent known to lack theactivity of an agent being tested, such as an antibody.

As used herein, “delaying development of a disease” means to defer,hinder, slow, retard, stabilize, suppress and/or postpone development ofthe disease (such as cancer). This delay can be of varying lengths oftime, depending on the history of the disease and/or individual beingtreated. As is evident to one skilled in the art, a sufficient orsignificant delay can, in effect, encompass prevention, in that theindividual does not develop the disease. For example, a late stagecancer, such as development of metastasis, may be delayed.

“Preventing,” as used herein, includes providing prophylaxis withrespect to the occurrence or recurrence of a disease in a subject thatmay be predisposed to the disease but has not yet been diagnosed withthe disease. Unless otherwise specified, the terms “reduce”, “inhibit”,or “prevent” do not denote or require complete prevention over all time,but just over the time period being measured.

A “therapeutically effective amount” of a substance/molecule, agonist orantagonist may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of thesubstance/molecule, agonist or antagonist to elicit a desired responsein the individual. A therapeutically effective amount is also one inwhich any toxic or detrimental effects of the substance/molecule,agonist or antagonist are outweighed by the therapeutically beneficialeffects. A therapeutically effective amount may be delivered in one ormore administrations. A therapeutically effective amount refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired therapeutic and/or prophylactic result.

The terms “pharmaceutical formulation” and “pharmaceutical composition”are used interchangeably and refer to a preparation which is in suchform as to permit the biological activity of the active ingredient(s) tobe effective, and which contains no additional components which areunacceptably toxic to a subject to which the formulation would beadministered. Such formulations may be sterile.

A “pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid, or liquid filler, diluent, encapsulating material,formulation auxiliary, or carrier conventional in the art for use with atherapeutic agent that together comprise a “pharmaceutical composition”for administration to a subject. A pharmaceutically acceptable carrieris non-toxic to recipients at the dosages and concentrations employedand are compatible with other ingredients of the formulation. Thepharmaceutically acceptable carrier is appropriate for the formulationemployed.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and sequential administrationin any order.

The term “concurrently” is used herein to refer to administration of twoor more therapeutic agents, where at least part of the administrationoverlaps in time, or where the administration of one therapeutic agentfalls within a short period of time relative to administration of theother therapeutic agent, or wherein the therapeutic effects of bothagents overlap for at least a period of time.

The term “sequentially” is used herein to refer to administration of twoor more therapeutic agents that does not overlap in time, or wherein thetherapeutic effects of the agents do not overlap.

As used herein, “in conjunction with” refers to administration of onetreatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modalitybefore, during, or after administration of the other treatment modalityto the individual.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

An “article of manufacture” is any manufacture (for example, a packageor container) or kit comprising at least one reagent, for example, amedicament for treatment of a disease or disorder (for example, cancer),or a probe for specifically detecting a biomarker described herein. Insome embodiments, the manufacture or kit is promoted, distributed, orsold as a unit for performing the methods described herein.

The terms “label” and “detectable label” mean a moiety attached, forexample, to an antibody or antigen to render a reaction (for example,binding) between the members of the specific binding pair, detectable.The labeled member of the specific binding pair is referred to as“detectably labeled.” Thus, the term “labeled binding protein” refers toa protein with a label incorporated that provides for the identificationof the binding protein. In some embodiments, the label is a detectablemarker that can produce a signal that is detectable by visual orinstrumental means, for example, incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (for example, streptavidin containing afluorescent marker or enzymatic activity that can be detected by opticalor colorimetric methods). Examples of labels for polypeptides include,but are not limited to, the following: radioisotopes or radionuclides(for example, ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho,or ¹⁵³Sm); chromogens, fluorescent labels (for example, FITC, rhodamine,lanthanide phosphors), enzymatic labels (for example, horseradishperoxidase, luciferase, alkaline phosphatase); chemiluminescent markers;biotinyl groups; predetermined polypeptide epitopes recognized by asecondary reporter (for example, leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags);and magnetic agents, such as gadolinium chelates. Representativeexamples of labels commonly employed for immunoassays include moietiesthat produce light, for example, acridinium compounds, and moieties thatproduce fluorescence, for example, fluorescein. In this regard, themoiety itself may not be detectably labeled but may become detectableupon reaction with yet another moiety.

Exemplary CD33-Binding Polypeptides

CD33-binding polypeptides are provided herein. In various embodiments,the CD33-binding polypeptides comprise at least one VHH domain thatbinds CD33. In some embodiments, the CD33 is a human CD33. In someembodiments, a CD33-binding polypeptide blocks binding of CD33 to asialic acid. In some embodiments, a CD33-binding polypeptide providedherein comprises one, two, three, four, five, six, seven, or eight VHHdomains that bind CD33. In some embodiments, a CD33-binding polypeptideprovided herein comprises one, two, three, or four VHH domains that bindCD33. CD33-binding polypeptides may comprise one or more VHH domainsthat bind one or more target proteins other than CD33. Such polypeptidesmay be referred to as “multispecific” polypeptides.

In some embodiments, a CD33-binding polypeptide comprises at least oneVHH domain that binds CD33 and an Fc region. In some embodiments, aCD33-binding polypeptide provided herein comprises one, two, three, orfour VHH domains and an Fc region. In some embodiments, an Fc regionmediates dimerization of the CD33-binding polypeptide at physiologicalconditions such that a dimer is formed that doubles the number of CD33binding sites. For example, a CD33-binding polypeptide comprising threeVHH domains that bind CD33 and an Fc region is trivalent as a monomer,but at physiological conditions, the Fc region may mediate dimerization,such that the CD33-binding polypeptide exists as a hexavalent dimerunder such conditions.

In some embodiments, a CD33-binding polypeptide comprises at least twoVHH domains, wherein a first VHH domain binds a first epitope of CD33and a second VHH domain binds a second epitope of CD33. When theCD33-binding polypeptide comprises a VHH domain that binds a firstepitope of CD33 and a VHH domain that binds a second epitope of CD33,the CD33-binding polypeptide may be referred to as “biepitopic” or“bispecific.” In some embodiments, a CD33-binding polypeptide comprisesat least two VHH domains, wherein a first VHH domain binds CD33 and asecond VHH domain binds an antigen other than CD33. Such polypeptidesmay be referred to as “bispecific” or “multispecific.”

Nonlimiting exemplary CD33-binding polypeptides are shown in Table 2.The sequences for the indicated single-domain antibodies are shown inthe Table of Certain Sequences herein. A polypeptide name that beginswith “hz” indicates that it is a humanized version of the correspondingparental polypeptide.

TABLE 2 Polypeptides comprising at least one VHH that binds CD33 VHH (orKP version or Name CDRs SS version) A07 SEQ ID NOs: 3, 4, and 5 SEQ IDNO: 2 (or 123) 401-A9 SEQ ID NOs: 7, 8, and 9 SEQ ID NO: 6 (or 131) B07SEQ ID NOs: 11, 12, and 13 SEQ ID NO: 10 (or 124) 1C7 SEQ ID NOs: 15,16, and 17 SEQ ID NO: 14 (or 125) 1E4 SEQ ID NOs: 19, 20, and 21 SEQ IDNO: 18 (or 126) F02 SEQ ID NOs: 23, 24, and 25 SEQ ID NO: 22 (or 127)1G3 SEQ ID NOs: 27, 28, and 29 SEQ ID NO: 26 (or 128) G11 SEQ ID NOs:31, 32, and 33 SEQ ID NO: 30 (or 129) 1H9 SEQ ID NOs: 35, 36, and 37 SEQID NO: 34 (or 130) hzA07v4 SEQ ID NOs: 47, 48, and 49 SEQ ID NO: 38 (or114) hzB07v7 SEQ ID NOs: 50, 51, and 52 SEQ ID NO: 39 (or 115) hz1C7v1SEQ ID NOs: 53, 54, and 55 SEQ ID NO: 40 (or 116) hz1C7v11 SEQ ID NOs:56, 57, and 58 SEQ ID NO: 41 (or 117) hz1E4v2 SEQ ID NOs: 59, 60, and 61SEQ ID NO: 42 (or 118) hzF02v18 SEQ ID NOs: 62, 63, and 64 SEQ ID NO: 43(or 119) hz1G3v3 SEQ ID NOs: 65, 66, and 67 SEQ ID NO: 44 (or 120)hzG11v2 SEQ ID NOs: 68, 69, and 70 SEQ ID NO: 45 (or 121) hz1H9v2 SEQ IDNOs: 71, 72, and 73 SEQ ID NO: 46 (or 122)

CD33-Binding Polypeptides

In various embodiments, a VHH domain that binds CD33 comprises a CDR1sequence selected from SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 47,50, 53, 56, 59, 62, 65, 68, and 71; a CDR2 sequence selected from SEQ IDNOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 48, 51, 54, 57, 60, 63, 66, 69,and 72; and a CDR3 sequence selected from SEQ ID NOs: 5, 9, 13, 17, 21,25, 29, 33, 37, 49, 52, 55, 58, 61, 64, 67, 70, and 73. In variousembodiments, a VHH domain that binds CD33 comprises CDR1, CDR2, and CDR3sequences selected from: SEQ ID NOs: 3, 4, and 5; SEQ ID NOs: 7, 8, and9; SEQ ID NOs: 11, 12, and 13; SEQ ID NOs: 15, 16, and 17; SEQ ID NOs:19, 20, and 21; SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 27, 28, and 29;SEQ ID NOs: 31, 32, and 33; SEQ ID NOs: 35, 36, and 37; SEQ ID NOs: 47,48, and 49; SEQ ID NOs: 50, 51, and 52; SEQ ID NOs: 53, 54, and 55; SEQID NOs: 56, 57, and 58; SEQ ID NOs: 59, 60, and 61; SEQ ID NOs: 62, 63,and 64; SEQ ID NOs: 65, 66, and 67; SEQ ID NOs: 68, 69, and 70; and SEQID NOs: 71, 72, and 73. In various embodiments, the VHH domain ishumanized.

In some embodiments, a VHH domain that binds CD33 comprises an aminoacid sequence that is at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% identical to an amino acidsequence selected from SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38,39, 40, 41, 42, 43, 44, 45, 46, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, and 131. In someembodiments, a VHH domain that binds CD33 comprises an amino acidsequence selected from SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38,39, 40, 41, 42, 43, 44, 45, 46, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, and 131.

In various embodiments, a CD33-binding polypeptide comprises one, two,three, or four VHH domains that bind CD33.

In various embodiments, CD33-binding polypeptide comprises at least oneVHH domain that binds CD33 and at least one VHH domain that binds anatural killer cell antigen or a T-cell antigen. In some suchembodiments, the CD33-binding polypeptide may be referred to as amultispecific antibody.

In some embodiments, a CD33 binding polypeptide comprises at least oneVHH domain described herein fused to an Fc region. In some embodiments,the Fc region has a sequence selected from SEQ ID NOs: 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, and 109.

In some embodiments, a VHH domain that binds CD33 may be humanized.Humanized antibodies (such as sdAbs or VHH-containing polypeptides) areuseful as therapeutic molecules because humanized antibodies reduce oreliminate the human immune response to non-human antibodies, which canresult in an immune response to an antibody therapeutic, and decreasedeffectiveness of the therapeutic. Generally, a humanized antibodycomprises one or more variable domains in which CDRs, (or portionsthereof) are derived from a non-human antibody, and FRs (or portionsthereof) are derived from human antibody sequences. A humanized antibodyoptionally will also comprise at least a portion of a human constantregion. In some embodiments, some FR residues in a humanized antibodyare substituted with corresponding residues from a non-human antibody(for example, the antibody from which the CDR residues are derived), forexample, to restore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, forexample, in Almagro and Fransson, (2008) Front. Biosci. 13: 1619-1633,and are further described, for example, in Riechmann et al., (1988)Nature 332:323-329; Queen et al., (1989) Proc. Natl Acad. Sci. USA 86:10029-10033; U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and7,087,409; Kashmiri et al., (2005) Methods 36:25-34; Padlan, (1991) Mol.Immunol. 28:489-498 (describing “resurfacing”); Dall'Acqua et al.,(2005) Methods 36:43-60 (describing “FR shuffling”); and Osbourn et al.,(2005) Methods 36:61-68 and Klimka et al., (2000) Br. J. Cancer,83:252-260 (describing the “guided selection” approach to FR shuffling).

Human framework regions that can be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, for example, Sims et al. (1993) J. Immunol. 151:2296);framework regions derived from the consensus sequence of humanantibodies of a particular subgroup of heavy chain variable regions(see, for example, Carter et al. (1992) Proc. Natl. Acad. Sci. USA,89:4285; and Presta et al. (1993) J. Immunol, 151:2623); human mature(somatically mutated) framework regions or human germline frameworkregions (see, for example, Almagro and Fransson, (2008) Front. Biosci.13:1619-1633); and framework regions derived from screening FR libraries(see, for example, Baca et al., (1997) J Biol. Chem. 272: 10678-10684and Rosok et al., (1996) J. Biol. Chem. 271:22611-22618). Typically, theFR regions of a VHH are replaced with human FR regions to make ahumanized VHH. In some embodiments, certain FR residues of the human FRare replaced in order to improve one or more properties of the humanizedVHH. VHH domains with such replaced residues are still referred toherein as “humanized.”

In various embodiments, an Fc region included in a CD33-bindingpolypeptide is a human Fc region, or is derived from a human Fc region.

In some embodiments, an Fc region included in a CD33-binding polypeptideis derived from a human Fc region, and comprises a three amino aciddeletion in the lower hinge corresponding to IgG1 E233, L234, and L235,herein referred to as “Fe xELL” Fc xELL polypeptides do not engage FcγRsand thus are referred to as “effector silent” or “effector null”,however in some embodiments, xELL Fc regions bind FcRn and thereforehave extended half-life and transcytosis associated with FcRn mediatedrecycling.

In some embodiments, the Fc region included in a CD33-bindingpolypeptide is derived from a human Fc region and comprises mutationsM252Y and M428V, herein referred to as “Fc-YV”. In some embodiments,such mutations enhance binding to FcRn at the acidic pH of the endosome(near 6.5), while losing detectable binding at neutral pH (about 7.2),allowing for enhanced FcRn mediated recycling and extended half-life.

In some embodiments, the Fc region included in a CD33-bindingpolypeptide is derived from a human Fc region and comprises mutationsdesigned for heterodimerization, herein referred to as “knob” and“hole”. In some embodiments, the “knob” Fc region comprises the mutationT366W. In some embodiments, the “hole” Fc region comprises mutationsT366S, L368A, and Y407V. In some embodiments, Fc regions used forheterodimerization comprise additional mutations, such as the mutationS354C on a first member of a heterodimeric Fc pair that forms anasymmetric disulfide with a corresponding mutation Y349C on the secondmember of a heterodimeric Fc pair. In some embodiments, one member of aheterodimeric Fc pair comprises the modification H435R or H435K toprevent protein A binding while maintaining FcRn binding. In someembodiments, one member of a heterodimeric Fc pair comprises themodification H435R or H435K, while the second member of theheterodimeric Fc pair is not modified at H435. In various embodiments,the hold Fc region comprises the modification H435R or H435K (referredto as “hole-R” in some instances when the modification is H435R), whilethe knob Fc region does not. In some instances, the hole-R mutationimproves purification of the heterodimer over homodimeric hole Fcregions that may be present.

Nonlimiting exemplary Fc regions that may be used in a CD33-bindingpolypeptide include Fc regions comprising the amino acid sequences ofSEQ ID NOs: 74 to 109.

Chimeric Receptors and Engineered Cells

Provided herein are chimeric antigen receptors (CARs) having anextracellular domain comprising one or more of the CD33-binding VHHdomains provided herein. CAR constructs provided herein include anextracellular domain containing the one or more CD33-binding VHH domain,a transmembrane domain and an intracellular signaling region. The one ormore CD33-binding VHH domain which form the antigen binding unit of theCAR binds or is capable of binding, i.e. targets, CD33-binding withsufficient affinity such the CAR is useful in therapy in targeting acell or tissue expressing CD33-binding.

CARs are synthetic receptors typically containing an extracellulartargeting/binding moiety that is associated with one or more signalingdomains in a single fusion molecule that is expressed on the surface ofa cell, such as a T cell. Thus, CARs combine antigen-specificity and Tcell activating properties in a single fusion molecule. First generationCARs typically included the cytoplasmic region of the CD3zeta or Fc 1receptor γ chain as their signaling domain. First generation CARs havebeen tested in phase I clinical studies in patients with ovarian cancer,renal cancer, lymphoma, and neuroblastoma, where they have inducedmodest responses (reviewed in Sadelain et al., Curr Opin Immunol, 21(2): 215-223, 2009). Second generation CARs, which contain the signalingdomains of a costimulatory molecule, such as CD28, and CD3zeta, providedual signaling to direct combined activating and co-stimulatory signals.Third generation CARs are more complex with three or more signalingdomains (reviewed in Sadelain et al., Cancer Discovery (3), 388-398,2013 and Dotti et al, Immuno. Rev, 257 (1), 1-36, 2014).

In some embodiments, a provided CAR comprises a CD33-binding VHH domain.In some embodiments, the CAR contains at least two VHH domains thattarget one or more antigen. In one embodiment, the antigen bindingdomain of a CAR comprises two or at least two CD33-binding VHH domains,thus providing a bivalent molecule. In one embodiment, the antigenbinding domain comprises two or at least two CD33-binding VHH domains,but bind to different epitopes on CD33. In such cases, the antigenbinding domain comprises a first CD33-binding VHH domain that binds to afirst epitope of CD33 and a second VHH domain that binds to a secondepitope of CD33. The epitopes may be overlapping. Thus, in someembodiments, the antigen binding domain is biparatopic and the CAR is abiparatopic CAR. In yet another embodiment, the antigen binding domaincomprises two CD33-binding VHH domains that bind to the same epitopes onCD33.

The transmembrane domain of a CAR provided herein is a domain thattypically crosses or is capable of crossing or spanning the plasmamembrane and is connected, directly or indirectly (e.g. via a spacer,such as an immunoglobulin hinge sequence) to the extracellular antigenbinding domain and the endoplasmic portion containing the intracellularsignaling domain. In one embodiment, the transmembrane domain of the CARis a transmembrane region of a transmembrane protein (for example Type Itransmembrane proteins), an artificial hydrophobic sequence or acombination thereof. In one embodiment, the transmembrane domaincomprises the CD3zeta domain or CD28 transmembrane domain. Othertransmembrane domains will be apparent to those of skill in the art andmay be used in connection with embodiments of a CAR provided herein.

The intracellular signaling region of a CAR provided herein contains oneor more intracellular signaling domain that transmits a signal to a Tcell upon engagement of the antigen binding domain of the CAR, such asupon binding antigen. In some embodiments, the intracellular regioncontains an intracellular signaling domain that is or contains an ITAMsignaling domain. Exemplary intracellular signaling domains include, forexample, a signaling domain derived from chain of the T-cell receptorcomplex or any of its homologs (e.g., η chain, FcsRIy and β chains, MB 1(Iga) chain, B29 (Ig) chain, etc.), human CD3zeta chain, CD3polypeptides (Δ, δ and ε), syk family tyrosine kinases (Syk, ZAP 70,etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) and othermolecules involved in T-cell transduction, such as CD2, CD5, OX40 andCD28. In particular embodiments, the intracellular signaling regioncontains an intracellular signaling domain derived from the human CD3zeta chain.

In some embodiments, the intracellular signaling region of a CAR canfurther contain an intracellular signaling domain derived from acostimulatory molecule. In such examples, such a signaling domain mayenhance CAR-T cell activity, such as via enhancement of proliferation,survival and/or development of memory cells, after antigen specificengagement, for example, compared to a CAR that only contains an ITAMcontaining signaling domain, e.g. CD3 zeta. In some embodiments, theco-stimulatory domain is a functional signaling domain obtained from aprotein selected from: CD28, CD137 (4-IBB), CD134 (OX40), Dap10, CD27,CD2, CD5, ICAM-1, LFA-1 (CD11a/CD18), Lck, TNFR-I, TNFR-II, Fas, CD30,CD40 or combinations thereof. In particular embodiments, thecostimulatory signaling domain is derived or obtained from a humanprotein. In some aspects, the costimulatory signaling domain is derivedor obtained from human CD28 or human CD137 (4-IBB).

In some embodiments, the costimulatory signaling domain is a derivedfrom CD28 or 41BB.

In particular embodiments, the CAR further comprises a hinge or spacerregion which connects the extracellular antigen binding domain and thetransmembrane domain. This hinge or spacer region can be used to achievedifferent lengths and flexibility of the resulting CAR. Examples of thea hinge or spacer region that can be used include, but are not limitedto, Fc fragments of antibodies or fragments or derivatives thereof,hinge regions of antibodies, or fragments or derivatives thereof, CH2regions of antibodies, CH3 regions of antibodies, artificial spacersequences, for example peptide sequences, or combinations thereof. Otherhinge or spacer region will be apparent to those of skill in the art andmay be used. In one embodiment, the hinge is an lgG4 hinge or a CD8Ahinge.

In some embodiments, the spacer and transmembrane domain are the hingeand transmembrane domain derived from CD8.

Also provided herein is an isolated nucleic acid construct comprising atleast one nucleic acid encoding a CAR as provided herein. In someaspects, the construct is an expression vector for expression of the CARin a cell. The expression vector may be a viral vector. Viral vectortechnology is well known in the art and is described, for example, inSambrook et al. (Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York, 2013). A number of viral based systems havebeen developed for gene transfer into mammalian cells. For example,retroviruses such as, adenovirus vectors are used. In one embodiment, alentivirus vector is used.

In a further aspect, also provided is an isolated cell or cellpopulation comprising one or more nucleic acid construct as describedabove. Also provided is an isolated cell or cell population that hasbeen genetically modified to express a CAR provided herein. Thus,provided herein are genetically engineered cells which comprise, such asstably express, a CAR provided herein. In one embodiment, the cell isselected from the group consisting of a T cell, a Natural Killer (NK)cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, hematopoieticstem cells and/or pluripotent embryonic/induced stem cells. In somecases, the cell is a T cell, such as a CD4 and/or CD8 T cell. In someembodiments, the cells are autologous to the subject. For example, insome embodiments, T cells may be isolated from a patient (also calledprimary T cells) for engineering, e.g. transfection or transduction,with a CAR nucleic acid construct.

In an exemplary example, primary T-cells can be purified ex vivo (CD4cells or CD8 cells or both) and stimulated with a TCR/CD28 agonists,such as anti-CD3/anti-CD28 coated beads. After a 2 or 3 day activationprocess, a recombinant expression vector encoding the CAR can be stablyintroduced into the primary T cells through standard lentiviral orretroviral transduction protocols or plasmid electroporation strategies.Cells can be monitored for CAR expression by, for example, flowcytometry using anti-epitope tag or antibodies that cross-react withnative parental molecule. T-cells that express the CAR can be enrichedthrough sorting with anti-epitope tag antibodies or enriched for high orlow expression depending on the application.

The CAR engineered T-cells can be assayed for appropriate function by avariety of means. In some cases, in vitro cytotoxicity, proliferation,or cytokine assays (e.g., IFN-gamma expression) can be used to assessthe function of engineered T-cells. Exemplary standard endpoints arepercent lysis of a tumor line, proliferation of the engineered T-cell,or IFN-gamma protein expression in culture supernatant. In some cases,the ability to stimulate activation of T cells upon stimulation of theCAR, e.g. via antigen, can be assessed, such as by monitoring expressionof activation markers such as CD69, CD44, or CD62L, proliferation and/orcytokine production.

Polypeptide Expression and Production

Nucleic acid molecules comprising polynucleotides that encode aCD33-binding polypeptide are provided. In some embodiments, the nucleicacid molecule may also encode a leader sequence that directs secretionof the CD33-binding polypeptide, which leader sequence is typicallycleaved such that it is not present in the secreted polypeptide. Theleader sequence may be a native heavy chain (or VHH) leader sequence, ormay be another heterologous leader sequence.

Nucleic acid molecules can be constructed using recombinant DNAtechniques conventional in the art. In some embodiments, a nucleic acidmolecule is an expression vector that is suitable for expression in aselected host cell.

Vectors comprising nucleic acids that encode the CD33-bindingpolypeptides described herein are provided. Such vectors include, butare not limited to, DNA vectors, phage vectors, viral vectors,retroviral vectors, etc. In some embodiments, a vector is selected thatis optimized for expression of polypeptides in a desired cell type, suchas CHO or CHO-derived cells, or in NSO cells. Exemplary such vectors aredescribed, for example, in Running Deer et al., Biotechnol. Prog.20:880-889 (2004).

In some embodiments, a CD33-binding polypeptide may be expressed inprokaryotic cells, such as bacterial cells; or in eukaryotic cells, suchas fungal cells (such as yeast), plant cells, insect cells, andmammalian cells. Such expression may be carried out, for example,according to procedures known in the art. Exemplary eukaryotic cellsthat may be used to express polypeptides include, but are not limitedto, COS cells, including COS 7 cells; 293 cells, including 293-6E cells;CHO cells, including CHO-S, DG44. Lec13 CHO cells, and FUT8 CHO cells;PER.C6® cells (Crucell); and NSO cells. In some embodiments, theCD33-binding polypeptides may be expressed in yeast. See, e.g., U.S.Publication No. US 2006/0270045 A1. In some embodiments, a particulareukaryotic host cell is selected based on its ability to make desiredpost-translational modifications to the polypeptide. For example, insome embodiments, CHO cells produce polypeptides that have a higherlevel of sialylation than the same polypeptide produced in 293 cells.

Introduction of one or more nucleic acids (such as vectors) into adesired host cell may be accomplished by any method, including but notlimited to, calcium phosphate transfection, DEAE-dextran mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection, etc. Nonlimiting exemplary methods aredescribed, for example, in Sambrook et al., Molecular Cloning, ALaboratory Manual, 3^(rd) ed. Cold Spring Harbor Laboratory Press(2001). Nucleic acids may be transiently or stably transfected in thedesired host cells, according to any suitable method.

Host cells comprising any of the nucleic acids or vectors describedherein are also provided. In some embodiments, a host cell thatexpresses a CD33-binding polypeptide described herein is provided. TheCD33-binding polypeptides expressed in host cells can be purified by anysuitable method. Such methods include, but are not limited to, the useof affinity matrices or hydrophobic interaction chromatography. Suitableaffinity ligands include the ROR1 ECD and agents that bind Fc regions.For example, a Protein A, Protein G, Protein A/G, or an antibodyaffinity column may be used to bind the Fc region and to purify aCD33-binding polypeptide that comprises an Fc region. Hydrophobicinteractive chromatography, for example, a butyl or phenyl column, mayalso suitable for purifying some polypeptides such as antibodies. Ionexchange chromatography (for example anion exchange chromatographyand/or cation exchange chromatography) may also suitable for purifyingsome polypeptides such as antibodies. Mixed-mode chromatography (forexample reversed phase/anion exchange, reversed phase/cation exchange,hydrophilic interaction/anion exchange, hydrophilic interaction/cationexchange, etc.) may also suitable for purifying some polypeptides suchas antibodies. Many methods of purifying polypeptides are known in theart.

In some embodiments, the CD33-binding polypeptide is produced in acell-free system. Nonlimiting exemplary cell-free systems are described,for example, in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009);Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol.Adv. 21: 695-713 (2003).

In some embodiments, CD33-binding polypeptides prepared by the methodsdescribed above are provided. In some embodiments, the CD33-bindingpolypeptide is prepared in a host cell. In some embodiments, theCD33-binding polypeptide is prepared in a cell-free system. In someembodiments, the CD33-binding polypeptide is purified. In someembodiments, a cell culture media comprising a CD33-binding polypeptideis provided.

In some embodiments, compositions comprising antibodies prepared by themethods described above are provided. In some embodiments, thecomposition comprises a CD33-binding polypeptide prepared in a hostcell. In some embodiments, the composition comprises a CD33-bindingpolypeptide prepared in a cell-free system. In some embodiments, thecomposition comprises a purified CD33-binding polypeptide.

Exemplary Methods of Treating Diseases Using CD33-Binding Polypeptides

In some embodiments, methods of treating disease in an individualcomprising administering a CD33-binding polypeptide or cells expressinga CD33-binding polypeptide are provided. In some embodiments, methodsfor treating cancer in an individual are provided. In some embodiments,methods for treating CD33-expressing or CD33-positive cancer in anindividual are provided. The method comprises administering to theindividual an effective amount of a CD33-binding polypeptide or cellsexpressing a CD33-binding polypeptide provided herein. In someembodiments, the CD33-binding polypeptide blocks binding of CD33 to asialic acid. In some embodiments, the CD33-binding polypeptide is usedto bring a cytotoxic agent to a CD33-expressing cell. In some suchembodiments, the CD33-binding polypeptide comprises a binding domainthat binds a cytotoxic T cell or NK cell. In some such embodiments, thebinding domain binds CD3, T-cell receptor (TCR) α, TCRβ, CD28, CD16,CD32A, CD64, CD89, NKp46, or NKG2D. The binding domain may be, in someembodiments, a VHH domain or an antibody binding domain comprising aheavy chain variable region and a light chain variable region, such as aVH/VL, scFv, Fab fragment, etc.

In some embodiments, the CD33-binding polypeptide is linked to acytotoxic agent to form an immunoconjugate. Various cytotoxic agentsused in immunoconjugates are known in the art, and include, but are notlimited to, calicheamicins, auristatins, dolastatins, tubulicins,maytansinoids, cryptophycins, duocarmycins, esperamicins,pyrrolobenzodiazepines, and enediyne antibiotics.

In some embodiments, the CD33-binding polypeptide is a chimeric antigenreceptor expressed on a cytotoxic cell, such as a T cell (CAR-T) or NKcell (CAR-NK). Such methods of treatment may be in humans or animals. Insome embodiments, methods of treating humans are provided.

Nonlimiting exemplary cancers that may be treated with CD33-bindingpolypeptides or cells expressing CD33-binding polypeptides providedherein include, but are not limited to, lymphoma; Hodgkin's lymphoma;non-Hodgkin's lymphoma; B-cell lymphoma; low grade/follicularnon-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; Waldenstrom's macroglobulinemia; acute myeloidleukemia (AML); chronic lymphocytic leukemia (CLL); acute lymphoblasticleukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia. Insome embodiments, the cancer is a CD33-expessing (i.e., CD33-positive)cancer.

The CD33-binding polypeptides or cells expressing CD33-bindingpolypeptides can be administered as needed to subjects. Determination ofthe frequency of administration can be made by persons skilled in theart, such as an attending physician based on considerations of thecondition being treated, age of the subject being treated, severity ofthe condition being treated, general state of health of the subjectbeing treated and the like. In some embodiments, an effective dose of aCD33-binding polypeptides or cells expressing CD33-binding polypeptidesis administered to a subject one or more times. In some embodiments, aneffective dose of a CD33-binding polypeptides or cells expressingCD33-binding polypeptides is administered to the subject daily,semiweekly, weekly, every two weeks, once a month, etc. An effectivedose of a CD33-binding polypeptides or cells expressing CD33-bindingpolypeptides is administered to the subject at least once. In someembodiments, the effective dose of a CD33-binding polypeptide or cellsexpressing a CD33-binding polypeptide may be administered multipletimes, including multiple times over the course of at least a month, atleast six months, or at least a year.

In some embodiments, pharmaceutical compositions are administered in anamount effective for treating (including prophylaxis of) cancer. Thetherapeutically effective amount is typically dependent on the weight ofthe subject being treated, his or her physical or health condition, theextensiveness of the condition to be treated, or the age of the subjectbeing treated. In general, antibodies may be administered in an amountin the range of about 0.05 mg/kg body weight to about 100 mg/kg bodyweight per dose. In some embodiments, antibodies may be administered inan amount in the range of about 10 μg/kg body weight to about 100 mg/kgbody weight per dose. In some embodiments, antibodies may beadministered in an amount in the range of about 50 μg/kg body weight toabout 5 mg/kg body weight per dose. In some embodiments, antibodies maybe administered in an amount in the range of about 100 μg/kg body weightto about 10 mg/kg body weight per dose. In some embodiments, antibodiesmay be administered in an amount in the range of about 100 μg/kg bodyweight to about 20 mg/kg body weight per dose. In some embodiments,antibodies may be administered in an amount in the range of about 0.5mg/kg body weight to about 20 mg/kg body weight per dose. In someembodiments, antibodies may be administered in an amount in the range ofabout 0.5 mg/kg body weight to about 10 mg/kg body weight per dose. Insome embodiments, antibodies may be administered in an amount in therange of about 0.05 mg/kg body weight to about 20 mg/kg body weight perdose. In some embodiments, antibodies may be administered in an amountin the range of about 0.05 mg/kg body weight to about 10 mg/kg bodyweight per dose. In some embodiments, antibodies may be administered inan amount in the range of about 5 mg/kg body weight or lower, forexample less than 4, less than 3, less than 2, or less than 1 mg/kg ofthe antibody.

In some embodiments, CD33-binding polypeptides or cells expressingCD33-binding polypeptides can be administered in vivo by various routes,including, but not limited to, intravenous, intra-arterial, parenteral,intraperitoneal or subcutaneous. The appropriate formulation and routeof administration may be selected according to the intended application.

In some embodiments, a therapeutic treatment using a CD33-bindingpolypeptide is achieved by targeting a cytotoxic agent to aCD33-expressing cell, such as a CD33-expressing cancer cell. In somesuch embodiments, the CD33-binding polypeptide is a chimeric antigenreceptor expressed on a cytotoxic cell, such as a T cell or NK cell.

Pharmaceutical Compositions

In some embodiments, compositions comprising CD33-binding polypeptidesare provided in formulations with a wide variety of pharmaceuticallyacceptable carriers (see, for example, Gennaro, Remington: The Scienceand Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus,20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and DrugDelivery Systems, 7^(th) ed., Lippencott Williams and Wilkins (2004);Kibbe et al., Handbook of Pharmaceutical Excipients, 3^(rd) ed.,Pharmaceutical Press (2000)). Various pharmaceutically acceptablecarriers, which include vehicles, adjuvants, and diluents, areavailable. Moreover, various pharmaceutically acceptable auxiliarysubstances, such as pH adjusting and buffering agents, tonicityadjusting agents, stabilizers, wetting agents and the like, are alsoavailable. Non-limiting exemplary carriers include saline, bufferedsaline, dextrose, water, glycerol, ethanol, and combinations thereof.

In some embodiments, a pharmaceutical composition comprises aCD33-binding polypeptide at a concentration of at least 10 mg/mL, 20mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90mg/mL, 100 mg/mL, 125 mg/mL, 150 mg/mL, 175 mg/mL, 200 mg/mL, 225 mg/mL,or 250 mg/mL.

Combination Therapy

CD33-binding polypeptides or engineered cells of the present disclosurecan be administered alone or in combination with other modes oftreatment, such as other anti-cancer agents. They can be providedbefore, substantially contemporaneous with, or after other modes oftreatment (i.e., concurrently or sequentially). In some embodiments, themethod of treatment described herein can further include administering:radiation therapy, chemotherapy, vaccination, targeted tumor therapy,CAR-T therapy, oncolytic virus therapy, cancer immunotherapy, cytokinetherapy, surgical resection, chromatin modification, ablation,cryotherapy, an antisense agent against a tumor target, a siRNA agentagainst a tumor target, a microRNA agent against a tumor target or ananti-cancer/tumor agent, or a biologic, such as an antibody, cytokine,or receptor extracellular domain-Fc fusion.

In some embodiments, a CD33-binding polypeptide provided herein is givenconcurrently with one or more chemotherapeutic agent, CAR-T (chimericantigen receptor T-cell) therapy, oncolytic virus therapy, cytokinetherapy, and/or agents that target other checkpoint molecules, such asVISTA, gpNMB, B7H4, HHLA2, CD73, CTLA4, TIGIT, etc.

In some embodiments, the CD33-binding polypeptide or engineered cells ofthe present disclosure is used in combination with other anti-tumoragents, such as anti-HER-2 antibodies, anti-CD20 antibodies, anepidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosinekinase inhibitor), HER1/EGFR inhibitor (e.g., erlotinib (TARCEVA®),platelet derived growth factor inhibitors (e.g., GLEEVEC® (ImatinibMesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons, CTLA4inhibitors (e.g., anti-CTLA antibody ipilimumab (YERVOY®)), PD-1inhibitors (e.g., anti-PD 1 antibodies, BMS-936558) PDL1 inhibitors(e.g., anti-PDL1 antibodies, MPDL3280A), PDL2 inhibitors (e.g.,anti-PDL2 antibodies), cytokines, antagonists (e.g., neutralizingantibodies) that bind to one or more of the following targets ErbB2,ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA, PD-1, PDL1, PDL2, CTLA4, orVEGF receptor(s), TRAIL/Apo2, and other bioactive and organic chemicalagents, etc.

In some embodiments, a CD33-binding polypeptide or engineered cellprovided herein is given concurrently with a PD-1/or PD-L1 therapy.Examples of PD-1/PD-L1 therapy include nivolumab (BMS); pidilizumab(CureTech, CT-011), pembrolizumab (Merck); durvalumab(Medimmune/AstraZeneca); atezolizumab (Genentech/Roche); avelumab(Pfizer); AMP-224 (Amplimmune); BMS-936559; AMP-514 (Amplimmune);MDX-1105 (Merck); TSR-042 (Tesaro/AnaptysBio, ANB-011); STI-A1010(Sorrento Therapeutics); STI-A1110 (Sorrento Therapeutics); and otheragents that are directed against programmed death-1 (PD-1) or programmeddeath ligand 1 (PD-L1).

In some embodiments, the CD33-binding polypeptide or engineered cell ofthe present disclosure may be used in combination with achemotherapeutic agent. Examples of chemotherapeutic agents include, butare not limited to, alkylating agents such as thiotepa and CYTOXAN®cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegaI1 (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluorometlhylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g.,erlotinib (TARCEVA®)) and VEGF-A that reduce cell proliferation andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

Further nonlimiting exemplary chemotherapeutic agents includeanti-hormonal agents that act to regulate or inhibit hormone action oncancers such as anti-estrogens and selective estrogen receptormodulators (SERMs), including, for example, tamoxifen (includingNOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and FARESTON® toremifene;aromatase inhibitors that inhibit the enzyme aromatase, which regulatesestrogen production in the adrenal glands, such as, for example,4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN®exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA®letrozole, and ARIMIDEX® anastrozole; and anti-androgens such asflutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as wellas troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisenseoligonucleotides, particularly those which inhibit expression of genesin signaling pathways implicated in abherant cell proliferation, suchas, for example, PKC-alpha, Ralf and H-Ras; ribozymes such as a VEGFexpression inhibitor (e.g., ANGIOZYME® ribozyme) and a HER2 expressioninhibitor; vaccines such as gene therapy vaccines, for example,ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN®(aldesleukin) rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX®GnRH agoninst; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

In some embodiments, the CD33-binding polypeptide and the additionalagent are formulated into a single therapeutic composition, and theCD33-binding polypeptide and additional agent are administeredsimultaneously. Alternatively, the CD33-binding polypeptide orengineered cell and the additional agent are separate from each other,e.g., each is formulated into a separate therapeutic composition, andthe CD33-binding polypeptide or engineered cell and the additional agentare administered simultaneously, or the CD33-binding polypeptide orengineered cell and the additional agent are administered at differenttimes during a treatment regimen. For example, the CD33-bindingpolypeptide or engineered cell is administered prior to theadministration of the additional agent, the CD33-binding polypeptide orengineered cell is administered subsequent to the administration of theadditional agent, or the CD33-binding polypeptide or engineered cell andthe additional agent are administered in an alternating fashion. TheCD33-binding polypeptide and additional agent may be administered insingle doses or in multiple doses.

In some embodiments, the CD33-binding polypeptide or engineered cell andthe additional agent(s) are administered simultaneously. For example,the CD33-binding polypeptide and the additional agent(s) can beformulated in a single composition or administered as two or moreseparate compositions. In some embodiments, the CD33-binding polypeptideor engineered cell and the additional agent(s) are administeredsequentially, or the CD33-binding polypeptide or engineered cell and theadditional agent are administered at different times during a treatmentregimen.

Nonlimiting Exemplary Methods of Diagnosis and Treatment

In some embodiments, the methods described herein are useful forevaluating a subject and/or a specimen from a subject (e.g. a cancerpatient). In some embodiments, evaluation is one or more of diagnosis,prognosis, and/or response to treatment.

In some embodiments, the methods described herein comprise evaluating apresence, absence, or level of a protein. In some embodiments, themethods described herein comprise evaluating a presence, absence, orlevel of expression of a nucleic acid. The compositions described hereinmay be used for these measurements. For example, in some embodiments,the methods described herein comprise contacting a specimen of the tumoror cells cultured from the tumor with a therapeutic agent as describedherein.

In some embodiments, the evaluation may direct treatment (includingtreatment with the antibodies described herein). In some embodiments,the evaluation may direct the use or withholding of adjuvant therapyafter resection. Adjuvant therapy, also called adjuvant care, istreatment that is given in addition to the primary, main or initialtreatment. By way of non-limiting example, adjuvant therapy may be anadditional treatment usually given after surgery where all detectabledisease has been removed, but where there remains a statistical risk ofrelapse due to occult disease. In some embodiments, the polypeptides areused as an adjuvant therapy in the treatment of a cancer. In someembodiments, the polypeptides are used as the sole adjuvant therapy inthe treatment of a cancer. In some embodiments, the polypeptidesdescribed herein are withheld as an adjuvant therapy in the treatment ofa cancer. For example, if a patient is unlikely to respond to anantibody described herein or will have a minimal response, treatment maynot be administered in the interest of quality of life and to avoidunnecessary toxicity from ineffective chemotherapies. In such cases,palliative care may be used.

In some embodiments the polypeptides are administered as a neoadjuvanttherapy prior to resection. In some embodiments, neoadjuvant therapyrefers to therapy to shrink and/or downgrade the tumor prior to anysurgery. In some embodiments, neoadjuvant therapy means chemotherapyadministered to cancer patients prior to surgery. In some embodiments,neoadjuvant therapy means an antibody is administered to cancer patientsprior to surgery. Types of cancers for which neoadjuvant chemotherapy iscommonly considered include, for example, breast, colorectal, ovarian,cervical, bladder, and lung. In some embodiments, the polypeptides areused as a neoadjuvant therapy in the treatment of a cancer. In someembodiments, the use is prior to resection.

In some embodiments, the tumor microenvironment contemplated in themethods described herein is one or more of: tumor vasculature;tumor-infiltrating lymphocytes; fibroblast reticular cells; endothelialprogenitor cells (EPC); cancer-associated fibroblasts; pericytes; otherstromal cells; components of the extracellular matrix (ECM); dendriticcells; antigen presenting cells; T-cells; regulatory T-cells;macrophages; other lymphoid cells; neutrophils; and other immune cellslocated proximal to a tumor.

Kits

Also provided are articles of manufacture and kits that include any ofCD33-binding polypeptides as described herein, and suitable packaging.In some embodiments, the invention includes a kit with (i) aCD33-binding polypeptide, and (ii) instructions for using the kit toadminister the CD33-binding polypeptide to an individual.

Suitable packaging for compositions described herein are known in theart, and include, for example, vials (e.g., sealed vials), vessels,ampules, bottles, jars, flexible packaging (e.g., sealed Mylar orplastic bags), and the like. These articles of manufacture may furtherbe sterilized and/or sealed. Also provided are unit dosage formscomprising the compositions described herein. These unit dosage formscan be stored in a suitable packaging in single or multiple unit dosagesand may also be further sterilized and sealed. Instructions supplied inthe kits of the invention are typically written instructions on a labelor package insert (e.g., a paper sheet included in the kit), butmachine-readable instructions (e.g., instructions carried on a magneticor optical storage disk) are also acceptable. The instructions relatingto the use of the antibodies generally include information as to dosage,dosing schedule, and route of administration for the intended treatmentor industrial use. The kit may further comprise a description ofselecting an individual suitable or treatment.

The containers may be unit doses, bulk packages (e.g., multi-dosepackages) or sub-unit doses. For example, kits may also be provided thatcontain sufficient dosages of molecules disclosed herein to provideeffective treatment for an individual for an extended period, such asabout any of a week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, ormore. Kits may also include multiple unit doses of molecules andinstructions for use and packaged in quantities sufficient for storageand use in pharmacies, for example, hospital pharmacies and compoundingpharmacies. In some embodiments, the kit includes a dry (e.g.,lyophilized) composition that can be reconstituted, resuspended, orrehydrated to form generally a stable aqueous suspension of antibody.

EXAMPLES

The examples discussed below are intended to be purely exemplary of theinvention and should not be considered to limit the invention in anyway. The examples are not intended to represent that the experimentsbelow are all or the only experiments performed. Efforts have been madeto ensure accuracy with respect to numbers used (for example, amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is average molecular weight, temperature is in degreesCentigrade, and pressure is at or near atmospheric.

Example 1: CD33 Single-Domain Antibodies

Single domain antibodies targeting human CD33 were generated viaimmunization of llamas and alpaca with a recombinant version of thehuman CD33 extracellular domain.

Following the development of specific anti-CD33 antibody titers,llama/alpaca peripheral blood mononuclear cells (PBMCs) were isolatedfrom 500 mL of blood from the immunized animal and total mRNA wasisolated using the Qiagen RNeasy Maxi Kit and subsequently converted tofirst strand cDNA using Thermo Superscript IV Reverse Transcriptase andoligo-dT priming. VHH sequences were specifically amplified via PCRusing the cDNA as template and cloned into a yeast surface displayvector as VHH-Fc-AGA2 fusion proteins.

Yeast libraries displaying the VHH-Fc-AGA2 fusion proteins were enrichedusing recombinant forms of the CD33 ECD via magnetic bead isolationfollowed by fluorescence activated cell sorting (FACS). Sorted yeastwere plated out and isolated colonies were picked into 96-well blocksand grown in media that switched the expression from surface displayedVHH-Fc to secretion into the media. Supernatants from the 96-well yeastsecretion cultures were applied to 293F cells transiently transfectedwith CD33 (CD33 positive) or untransfected 293F cells (CD33 negative),washed, treated with fluorophore labelled anti-human IgG1 Fc secondaryantibody, and analyzed by 96-well flow cytometry.

Nucleic acid sequences encoding VHHs that bound to CD33 positive cellsand not to CD33 negative cells were cloned in-frame with a human Fcencoding region into mammalian expression vectors, and expressed bytransient transfection in HEK293 Freestyle cells (293F cells) or CHOcells using polyethylenimine. Supernatant was collected after 3-7 days,secreted recombinant protein was purified by protein A chromatography,and concentration was calculated from the absorbance at 280 nm andextinction coefficient.

The epitopes of single domain antibodies (sdAbs) that comprise VHHdomains that bind CD33 were compared using Bio-Layer Interferometry. 7ug/mL of AviTag™-histidine tagged human CD33 was immobilized onstreptavidin coated capture sensors. 100 nM of one sdAb was then loadedonto the CD33 antigen and allowed to come to equilibrium. The sensorswere then transferred into 100 nM of a second sdAb. An increase in assaysignal represents binding, indicating that the second sdAb was targetingan epitope distinct from the epitope of the first sdAb.

Camelid-derived CD33 VHHs were humanized using the human VH3-23 germlineas scaffold. Camelid residues that contribute to solubility,specificity, stability and/or affinity remained unmodified. Furthermore,where possible and as needed amino acid sequences that posse potentialdevelopability liabilities were modified to mitigate this risk. Inaddition all humanized variants contained the Leu11Glu (L11E)modification, as described in US20160207981.

The results indicate that one epitope was found among humanized versionsof sdAbs 1E4, 1H9, 1G3, and 1C7. As shown in FIG. 1, hz1E4v2, hz1H9v2,hz1G3v3, and hz1C7v1 have a common epitope.

Example 2: Binding of Polypeptides to CD33

Binding of sdAbs to human CD33 was assessed by flow cytometry. Each sdAbcomprised a VHH domain, as indicated in Table 3 below, and a human IgG1xELL Fc region in which amino acids Glu233, Leu234, and Leu235,according to EU numbering, were deleted (SEQ ID NO: 75). MOLM-13 cellswere transiently transfected with a plasmid encoding a sequencecomprising “CD33M” (SEQ ID NO: 112) or a plasmid encoding a sequencecomprising a truncated version, “CD33m” (SEQ ID NO: 113). The sequenceencoded by the CD33M plasmid includes the leader sequence, fullextracellular domain, transmembrane domain, and cytoplasmic domain. Thesequence encoded by the CD33m plasmid includes the leader sequence, themembrane proximal IgC2 domain, and the transmembrane domain.Untransfected HEK 293 cells were used as CD33-negative cells. Each celltype was plated at 30,000 cells/well in FACS buffer (PBS 1% BSA, 0.1%NaN₃ pH 7.4) on a 96-well round-bottom plate. The sdAbs were diluted inFACS buffer in a 3-fold, 11 point serial dilution. The sdAb dilutionswere added to the plated cells, and assay plates were incubated for 30minutes at 4° C. After washing twice in 150 μL of FACS buffer, cells ineach well were resuspended in 100 μL of 1:2000 Alexa Fluor647-conjugated Anti-Human IgG secondary diluted in FACS buffer andincubated at 4° C. for 30 minutes. The cells were washed twice more,then bound antibody was detected by flow cytometry.

Flow cytometric analysis was performed on an Intellicyte iQue Plus andfluorescence was plotted as median fluorescence intensity. The apparentaffinity (K_(d), nM) was determined using one site binding non-linearregression in PRISM graph software.

As shown in FIGS. 2A to 2M, the tested sdAbs displayed CD33 binding anddid not bind untransfected cells that do not express CD33. The apparentbinding affinities are shown in Table 3 below.

TABLE 3 SEQ ID NO of VHH sdAb CD33 isoform Apparent K_(d) (nM) domain1E4 CD33M 0.05 18 hz1E4v2 CD33M 0.32 42 1H9 CD33M 0.14 34 hz1H9v2 CD33M0.27 46 1G3 CD33M 0.49 26 hz1G3v3 CD33M 1.60 44 1C7 CD33M 6.64 14hz1C7v1 CD33M 6.25 40 hz1C7v11 CD33M 5.45 41 A07 CD33M 53.50 2 A07 CD33m19.90 2 hzA07v4 CD33M 56.61 38 hzA07v4 CD33m 11.76 38 F02 CD33M 15.25 22F02 CD33m 5.15 22 hzF02v18 CD33M 14.83 43 hzF02v18 CD33m 9.13 43 hzB07v7CD33M 0.75 39 G11 CD33M 1.1 30 hzG11v2 CD33M 2.67 45

The disclosure may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting of the disclosure. Scope of the disclosure is thusindicated by the appended claims rather than by the foregoingdescription, and all changes that come within the meaning and range ofequivalency of the claims are therefore intended to be embraced herein.

Table of Certain Sequences SEQ ID NO Description Sequence   1Human CD33; DPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIImature form SRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQ   2 A07QVQLVQSGGGLVQAGGSLTLSCAASRSSGIDVMGWYRQAPGKERELVAEISGVGDTNYAASLADRFTVSRDNAKNTVYLQMKNLKPEDTAVYYCNAHSFLDLVGAW GQGTQVTV 123A07 KP QVQLVQSGGGLVQAGGSLTLSCAASRSSGIDVMGWYRQAPGKERELVAEISGVGDTNYAASLADRFTVSRDNAKNTVYLQMKNLKPEDTAVYYCNAHSFLDLVGAW GQGTQVTVKP  3A07 CDR1 RSSGIDVMG  4 A07 CDR2 EISGVGDTN  5 A07 CDR3 HSFLDLVGA  6 401-A9QVQLQQSGGGLVQAGGSLRLSCAAPGSINSINVMEWYRQAPGKERDLVAGITSDGDTNYVDSVPGRFTITRDNTMRTVDLQMNNLKADDTAVYYCRARDWGSLTDY WGQGTQVTV 131401-A9 KP QVQLQQSGGGLVQAGGSLRLSCAAPGSINSINVMEWYRQAPGKERDLVAGITSDGDTNYVDSVPGRFTITRDNTMRTVDLQMNNLKADDTAVYYCRARDWGSLTDY WGQGTQVTVKP  7401-A9 CDR1 GSINSINVME  8 401-A9 CDR2 GITSDGDTN  9 401-A9 CDR3 RDWGSLTDY10 B07 EVQLVQSGGGLVQTGGSLRLSCGASGRTISDYVVGWFRQAPGKERAFVAAISRYGTTYYAASVQGRFTISRDNPRNTVYLQVDSLRPEDTAVYFCAALQNDVRNNH SPTSYDYWGQGTQVTV124 B07 KP EVQLVQSGGGLVQTGGSLRLSCGASGRTISDYVVGWFRQAPGKERAFVAAISRYGTTYYAASVQGRFTISRDNPRNTVYLQVDSLRPEDTAVYFCAALQNDVRNNH SPTSYDYWGQGTQVTVKP 11 B07 CDR1 GRTISDYVVG  12 B07 CDR2 AISRYGTTY  13 B07 CDR3LQNDVRNNHSPTSYDY  14 1C7QVQLQQSGGGLVQAGGSLRLSCAASGRTFSGYIMGWFRQVPGKERELVARISGNNLSTEYGGSVKGRFTISRDSAKETMYLQMNSLKPEDTAIYYCAAEYDYSSGD FVYWGQGTQVTV 1251C7 KP QVQLQQSGGGLVQAGGSLRLSCAASGRTFSGYIMGWFRQVPGKERELVARISGNNLSTEYGGSVKGRFTISRDSAKETMYLQMNSLKPEDTAIYYCAAEYDYSSGD FVYWGQGTQVTVKP  151C7 CDR1 GRTFSGYIMG  16 1C7 CDR2 RISGNNLSTE  17 1C7 CDR3 EYDYSSGDFVY  181E4 QVQLQQSGGGSVQAGGSLRLSCVASGSGFSASLMSWHRQAPGSQRDLVASITRDGRANYVDSVKDRFTISRDNAKNTAYLQMDSLKPEDTAAYYCHAYSFDYPIRS YWGQGTQVTV 1261E4 KP QVQLQQSGGGSVQAGGSLRLSCVASGSGFSASLMSWHRQAPGSQRDLVASITRDGRANYVDSVKDRFTISRDNAKNTAYLQMDSLKPEDTAAYYCHAYSFDYPIRS YWGQGTQVTVKP  191E4 CDR1 GSGFSASLMS  20 1E4 CDR2 SITRDGRAN  21 1E4 CDR3 YSFDYPIRSY  22F02 QVQLVQSGGGLVQAGGSLRLSCAASGSISSYNVVGWYRQLSGNERGGRTMVAQINAYGDTNYANAVVGRFTISRDDAKNTVYLHMSNLKPEDTGVYYCNGQRMLEN YTYRDQSWGQGTQVTV127 F02 KP QVQLVQSGGGLVQAGGSLRLSCAASGSISSYNVVGWYRQLSGNERGGRTMVAQINAYGDTNYANAVVGRFTISRDDAKNTVYLHMSNLKPEDTGVYYCNGQRMLEN YTYRDQSWGQGTQVTVKP 23 F02 CDR1 GSISSYNVVG  24 F02 CDR2 QINAYGDTN  25 F02 CDR3QRMLENYTYRDQS  26 1G3EVQLQQSGGGEVQPGGSLRLSCEASGETFSSYAMGWFRQAPGKGREWVAAITTSGDTTYYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAAHRGGGVID YWGQGTQVTV 1281G3 KP EVQLQQSGGGEVQPGGSLRLSCEASGETFSSYAMGWFRQAPGKGREWVAAITTSGDTTYYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAAHRGGGVID YWGQGTQVTVKP  271G3 CDR1 GFTFSSYAMG  28 1G3 CDR2 AITTSGDTTY  29 1G3 CDR3 HRGGGVIDY  30G11 EVQLVQSGGGSVQVGGSLRLSCAASGSTLNIDHIGWYRQAPGKERELVGVISSGAGPNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYNCNAWIDYGSGLP QNYWGQGTQVTV 129G11 KP EVQLVQSGGGSVQVGGSLRLSCAASGSTLNIDHIGWYRQAPGKERELVGVISSGAGPNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYNCNAWIDYGSGLP QNYWGQGTQVTVKP  31G11 CDR1 GSTLNIDHIG  32 G11 CDR2 viSSGAGPN  33 G11 CDR3 WIDYGSGLPQNY  341H9 EVQLQQSGGAVVQPGGSLRLSCAASGSIFSISIMGWYRQAPGKERELVASTTSSGTTNYVDSVKGRFTASRDNAKNTVYLQMNSLKPDDTAIYHCHAYIATTTDRG YRGYWGQGTQVTV 1301H9 KP EVQLQQSGGAVVQPGGSLRLSCAASGSIFSISIMGWYRQAPGKERELVASTTSSGTTNYVDSVKGRFTASRDNAKNTVYLQMNSLKPDDTAIYHCHAYIATTTDRG YRGYWGQGTQVTVKP 35 1H9 CDR1 GSIFSISIMG  36 1H9 CDR2 STTSSGTTN  37 1H9 CDR3YIATTTDRGYRGY  38 hzA07v4EVQLVESGGGEVQPGGSLRLSCAASRSSGIDVMGWYRQAPGKERELVAEISGVGDTNYAASLADRFTVSRDNAKNTVYLQMSSLRAEDTAVYYCNAHSFLDLVGAW GQGTLVTV 114hzA07v4 KP EVQLVESGGGEVQPGGSLRLSCAASRSSGIDVMGWYRQAPGKERELVAEISGVGDTNYAASLADRFTVSRDNAKNTVYLQMSSLRAEDTAVYYCNAHSFLDLVGAW GQGTLVTVKP  39hzB07v7 EVQLVESGGGEVQPGGSLRLSCAASGRTISDYVVGWERQAPGKERAFVAAISRYGTTYYAASVQGRFTISRDNPRNTVYLQVDSLRAEDTAVYYCAALQNDVRNNH SPTSYDYWGQGTLVTV115 hzB07v7 KP EVQLVESGGGEVQPGGSLRLSCAASGRTISDYVVGWERQAPGKERAFVAAISRYGTTYYAASVQGRFTISRDNPRNTVYLQVDSLRAEDTAVYYCAALQNDVRNNH SPTSYDYWGQGTLVTVKP 40 hz1C7v1 EVQLVESGGGEVQPGGSLRLSCAASGRTFSGYIMGWFRQAPGKERELVARISGNNLSTEYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAAEYDYSSGD FVYWGQGTLVTV 116hz1C7v1 KP EVQLVESGGGEVQPGGSLRLSCAASGRTFSGYIMGWFRQAPGKERELVARISGNNLSTEYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAAEYDYSSGD FVYWGQGTLVTVKP  41hz1C7v11 EVQLVESGGGEVQPGGSLRLSCAASGRTFSGYIMGWFRQAPGKERELVARISGNNLATEYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAAEYDYSSGD FVYWGQGTLVTV 117hz1C7v11 KP EVQLVESGGGEVQPGGSLRLSCAASGRTFSGYIMGWFRQAPGKERELVARISGNNLATEYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAAEYDYSSGD FVYWGQGTLVTVKP  42hz1E4v2 EVQLVESGGGEVQPGGSLRLSCAASGSGFSASLMSWHRQAPGKQRDLVASITRDGRANYVESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCHAYSFDYPIRS YWGQGTLVTV 118hz1E4v2 KP EVQLVESGGGEVQPGGSLRLSCAASGSGFSASLMSWHRQAPGKQRDLVASITRDGRANYVESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCHAYSFDYPIRS YWGQGTLVTVKP  43hzF02v18 EVQLVESGGGEVQPGGSLRLSCAASGSISSYNVMGWYRQAPGKQRELVAQINAYGDTNYANAVVGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCNGQRMLENYTY RDQSWGQGTLVTV 119hzF02v18 KP EVQLVESGGGEVQPGGSLRLSCAASGSISSYNVMGWYRQAPGKQRELVAQINAYGDTNYANAVVGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCNGQRMLENYTY RDQSWGQGTLVTVKP 44 hz1G3v3 EVQLVESGGGEVQPGGSLRLSCAASGETFSSYAMGWFRQAPGKGREWVAAITTSGDTTYYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAAHRGGGVID YWGQGTLVTV 120hz1G3v3 EVQLVESGGGEVQPGGSLRLSCAASGETFSSYAMGWFRQAPGKGREWVAAITTSGDTTYYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAAHRGGGVID YWGQGTLVTVKP  45hzG11v2 EVQLVESGGGEVQPGGSLRLSCAASGSTLNIDHIGWYRQAPGKERELVGVISSGAGPNYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCNAWIDYGSGLP QNYWGQGTLVTV 121hzG11v2 KP EVQLVESGGGEVQPGGSLRLSCAASGSTLNIDHIGWYRQAPGKERELVGVISSGAGPNYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCNAWIDYGSGLP QNYWGQGTLVTVKP  46hz1H9v2 EVQLVESGGGEVQPGGSLRLSCAASGSIFSISIMGWYRQAPGKERELVASTTSSGTTNYVESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCHAYIATTTDRG YRGYWGQGTLVTV 122hz1H9v2 KP EVQLVESGGGEVQPGGSLRLSCAASGSIFSISIMGWYRQAPGKERELVASTTSSGTTNYVESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCHAYIATTTDRG YRGYWGQGTLVTVKP 47 CDR1 or RSSGIDVMG hzA07v4  48 CDR2 of EISGVGDTN hzA07v4  49 CDR3 ofHSFLDLVGA hzA07v4  50 CDR1 or GRTISDYVVG hzB07v7  51 CDR2 of AISRYGTTYhzB07v7  52 CDR3 of LQNDVRNNHSPTSYDY hzB07v7  53 CDR1 of GRTFSGYIMGhz1C7v1  54 CDR2 of RISGNNLSTE hz1C7v1  55 CDR3 of EYDYSSGDFVY hz1C7v1 56 CDR1 of GRTFSGYIMG hz1C7v11  57 CDR2 of RISGNNLATE hz1C7v11  58CDR3 of EYDYSSGDFVY hz1C7v11  59 CDR1 of GSGFSASLMS hz1E4v2  60 CDR2 ofSITRDGRAN hz1E4v2  61 CDR3 of YSFDYPIRSY hz1E4v2  62 CDR1 of GSISSYNVMGhzF02v18  63 CDR2 of QINAYGDTN hzF02v18  64 CDR3 of QRMLENYTYRDQShzF02v18  65 CDR1 of GFTFSSYAMG hz1G3v3  66 CDR2 of AITTSGDTTY hz1G3v3 67 CDR3 of HRGGGVIDY hz1G3v3  68 CDR1 of GSTLNIDHIG hzG11v2  69 CDR2 ofVISSGAGPN hzGl1v2  70 CDR3 of WIDYGSGLPQNY hzG11v2  71 CDR1 ofGSIFSISIMG hz1H9v2  72 CDR2 of STTSSGTTN hz1H9v2  73 CDR3 ofYIATTTDRGYRGY hz1H9v2  74 human IgG1DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Fc regionKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 75 human IgG1 DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNxELL Fc WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP regionAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK  76Fc region DKTHTCPPCP APELLGGPSV FLFPPKPKDT LYISRTPEVT M252Y andCVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY M428V (YV)RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK S354C T366WGQPREPQVYT LPPCRDELTK NQVSLWCLVK GFYPSDIAVE knobWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVVHE ALHNHYTQKS LSLSPGK  77 Fc regionDKTHTCPPCP APELLGGPSV FLFPPKPKDT LYISRTPEVT M252Y,CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY M428V,RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK H435FL (YVR)GQPREPQVCT LPPSRDELTK NQVSLSCAVK GFYPSDIAVE T366S,WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQG L368A,NVFSCSVVHE ALHNRYTQKS LSLSPGK Y407V hole  78 Fc regionDKTHTC PPCPAPGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE xELL H435RDPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVLHQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVYTLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPENNYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPGK  79Fc region DKTHTC PPCPAPGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE xELL M252YDPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL and M428VHQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY (YV)TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPENNYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVVH EALHNHYTQK SLSLSPGK  80Fc region DKTHTC PPCPAPGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE xELL M252YDPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL and M428LHQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY (YL)TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPENNYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVLH EALHNHYTQK SLSLSPGK  81Fc region DKTHTC PPCPAPGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE xELL M252Y,DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL M428L,HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY H435FL (YLR)TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPENNYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVLH EALHNRYTQK SLSLSPGK  82Fc region DKTHTC PPCPAPGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE xELL M252Y,DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL M428V,HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY H435FL (YVR)TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPENNYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVVH EALHNRYTQK SLSLSPGK  83Fc region DKTHTC PPCPAPGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE xELL S354CDPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL T366W knobHQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVYTLPPCRDELT KNQVSLWCLV KGFYPSDIAV EWESNGQPENNYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK  84Fc region DKTHTC PPCPAPGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE xELL H435RDPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL S354C T366WHQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY knobTLPPCRDELT KNQVSLWCLV KGFYPSDIAV EWESNGQPENNYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPGK  85Fc region DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNxELL M252Y WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPand M428V APIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWE(YV) S354C SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVVHEALHNHT366W knob YTQKSLSLSPGK  86 Fc regionDKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFN xELL M252YWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP and M428LAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWE (YL) S354CSNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNH T366W knobYTQKSLSLSPGK  87 Fc regionDKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFN xELL M252Y,WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP M428L,APIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWE H435R (YLR)SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNR S354C T366WYTQKSLSLSPGK knob  88 Fc regionDKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFN xELL M252Y,WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP M428V,APIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWE H435FL (YVR)SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVVHEALHNR S354C T366WYTQKSLSLSPGK knob  89 Fc regionDKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN xELL T366S,WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP L368A,APIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWE Y407V holeSNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK  90Fc region DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNxELL H435R, WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP T366S,APIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWE L368A,SNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNR Y407V holeYTQKSLSLSPGK  91 Fc regionDKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFN xELL M252YWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP and M428VAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWE (YV) T366S,SNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVVHEALHNH L368A,YTQKSLSLSPGK Y407V hole  92 Fc regionDKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFN xELL M252YWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP and M428LAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWE (YL) T366S,SNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVLHEALHNH L368A,YTQKSLSLSPGK Y407V hole  93 Fc regionDKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFN xELL M252Y,WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP M428L,APIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWE H435R (YLR)SNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVLHEALHNR T366S,YTQKSLSLSPGK L368A, Y407V hole  94 Fc regionDKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFN xELL M252Y,WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP M428V,APIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWE H435FL (YVR)SNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVVHEALHNR T366S,YTQKSLSLSPGK L368A, Y407V hole  95 Fc regionDKTHTCPPCP APELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE H435RDPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVLHQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVYTLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPENNYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPGK  96Fc region DKTHTCPPCP APELLGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHEM252Y and DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL M428V (YV)HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVYTLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPENNYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVVH EALHNHYTQK SLSLSPGK  97Fc region DKTHTCPPCP APELLGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHEM252Y and DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL M428L (YL)HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVYTLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPENNYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVLH EALHNHYTQK SLSLSPGK  98Fc region DKTHTCPPCP APELLGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE M252Y,DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL M428L,HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY H435R (YLR)TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPENNYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVLH EALHNRYTQK SLSLSPGK  99Fc region DKTHTCPPCP APELLGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE M252Y,DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL M428V,HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY H435R (YVR)TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPENNYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVVH EALHNRYTQK SLSLSPGK 100Fc region DKTHTCPPCP APELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHES354C T366W DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL knobHQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVYTLPPCRDELT KNQVSLWCLV KGFYPSDIAV EWESNGQPENNYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 101Fc region DKTHTCPPCP APELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHEH435R S354C DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL T366W knobHQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVYTLPPCRDELT KNQVSLWCLV KGFYPSDIAV EWESNGQPENNYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPGK 102Fc region DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVM252Y and KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKM428L (YL) ALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVS354C T366W EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEAL knobHNHYTQKSLSLSPGK 103 Fc regionDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEV M252Y,KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK M428L,ALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAV H435R (YLR)EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEAL S354C T366WHNRYTQKSLSLSPGK knob 104 Fc regionDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEV M252Y,KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK M428V,ALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAV H435R (YVR)EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVVHEAL S354C T366WHNRYTQKSLSLSPGK knob 105 Fc regionDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV T366SKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK L368A,ALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAV Y407V holeEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK106 Fc region DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVH435R KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK T366S,ALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAV L368A,EWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEAL Y407V holeHNRYTQKSLSLSPGK 107 Fc regionDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLYISRTPEVTCVVVDVSHEDPEV M252Y andKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK M428V (YV)ALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAV T366S,EWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVVHEAL L368A,HNHYTQKSLSLSPGK Y407V hole 108 Fc regionDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLYISRTPEVTCVVVDVSHEDPEV M252Y andKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK M428L (YL)ALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAV T366S,EWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVLHEAL L368A,HNHYTQKSLSLSPGK Y407V hole 109 Fc regionDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLYISRTPEVTCVVVDVSHEDPEV M252Y,KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK M428L,ALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAV H435R (YLR)EWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVLHEAL T366S,HNRYTQKSLSLSPGK L368A, Y407V hole 110 CD33 ECDDPNEWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRERLLGDPSRNNCSLSIVDARRRDNGSYFERMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVH 111 CD33 ECD-DPNEWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRD AviTag ™-SPVATNKLDQEVQEETQGRERLLGDPSRNNCSLSIVDARRRDNGSYFERMERG HisSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHGGTGGSGLNDIFEAQKIEWHEHH HHHH 112 CD33MDPNEWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRERLLGDPSRNNCSLSIVDARRRDNGSYFERMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTT 113 CD33mDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPG DGSGKQETRAGVVH

1. A polypeptide comprising at least one VHH domain that binds CD33 andthat comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:47, 3, 7, 11, 15, 19, 23, 27, 31, 35, 50, 53, 56, 59, 62, 65, 68, or 71;a CDR2 comprising the amino acid sequence of SEQ ID NO: 48, 4, 8, 12,16, 20, 24, 28, 32, 36, 51, 54, 57, 60, 63, 66, 69, or 72; and a CDR3comprising the amino acid sequence of SEQ ID NO: 49, 5, 9, 13, 17, 21,25, 29, 33, 37, 52, 55, 58, 61, 64, 67, 70, or
 73. 2. The polypeptide ofclaim 1, wherein at least one VHH domain comprises a CDR1 comprising theamino acid sequence of SEQ ID NO: 47 or 3; a CDR2 comprising the aminoacid sequence of SEQ ID NO: 48 or 4; and a CDR3 comprising the aminoacid sequence of SEQ ID NO: 49 or
 5. 3. The polypeptide of claim 1,wherein at least one VHH domain comprises a CDR1 comprising the aminoacid sequence of SEQ ID NO: 7; a CDR2 comprising the amino acid sequenceof SEQ ID NO: 8; and a CDR3 comprising the amino acid sequence of SEQ IDNO:
 9. 4. The polypeptide of claim 1, wherein at least one VHH domaincomprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 11 or50; a CDR2 comprising the amino acid sequence of SEQ ID NO: 12 or 51;and a CDR3 comprising the amino acid sequence of SEQ ID NO: 13 or
 52. 5.The polypeptide of claim 1, wherein at least one VHH domain comprises aCDR1 comprising the amino acid sequence of SEQ ID NO: 15, 53, or 56; aCDR2 comprising the amino acid sequence of SEQ ID NO: 16, 54, or 57; anda CDR3 comprising the amino acid sequence of SEQ ID NO: 17, 55, or 58.6. The polypeptide of claim 1, wherein at least one VHH domain comprisesa CDR1 comprising the amino acid sequence of SEQ ID NO: 19 or 59; a CDR2comprising the amino acid sequence of SEQ ID NO: 20 or 60; and a CDR3comprising the amino acid sequence of SEQ ID NO: 21 or
 61. 7. Thepolypeptide of claim 1, wherein at least one VHH domain comprises a CDR1comprising the amino acid sequence of SEQ ID NO: 23 or 62; a CDR2comprising the amino acid sequence of SEQ ID NO: 24 or 63; and a CDR3comprising the amino acid sequence of SEQ ID NO: 25 or
 64. 8. Thepolypeptide of claim 1, wherein at least one VHH domain comprises a CDR1comprising the amino acid sequence of SEQ ID NO: 27 or 65; a CDR2comprising the amino acid sequence of SEQ ID NO: 28 or 66; and a CDR3comprising the amino acid sequence of SEQ ID NO: 29 or
 67. 9. Thepolypeptide of claim 1, wherein at least one VHH domain comprises a CDR1comprising the amino acid sequence of SEQ ID NO: 31 or 68; a CDR2comprising the amino acid sequence of SEQ ID NO: 32 or 69; and a CDR3comprising the amino acid sequence of SEQ ID NO: 33 or
 70. 10. Thepolypeptide of claim 1, wherein at least one VHH domain comprises a CDR1comprising the amino acid sequence of SEQ ID NO: 35 or 71; a CDR2comprising the amino acid sequence of SEQ ID NO: 36 or 72; and a CDR3comprising the amino acid sequence of SEQ ID NO: 37 or
 73. 11. Thepolypeptide of claim 1, wherein at least one VHH domain comprises aCDR1, a CDR2, and a CDR3, respectively comprising the amino acidsequences of SEQ ID NOs: 47, 48, and 49; SEQ ID NOs: 3, 4, and 5; SEQ IDNOs: 7, 8, and 9; SEQ ID NOs: 11, 12, and 13; SEQ ID NOs: 15, 16, and17; SEQ ID NOs: 19, 20, and 21; SEQ ID NOs: 23, 24, and 25; SEQ ID NOs:27, 28, and 29; SEQ ID NOs: 31, 32, and 33; SEQ ID NOs: 35, 36, and 37;SEQ ID NOs: 50, 51, and 52; SEQ ID NOs: 53, 54, and 55; SEQ ID NOs: 56,57, and 58; SEQ ID NOs: 59, 60, and 61; SEQ ID NOs: 62, 63, and 64; SEQID NOs: 65, 66, and 67; SEQ ID NOs: 68, 69, and 70; or SEQ ID NOs: 71,72, and
 73. 12. The polypeptide of claim 1, wherein at least one VHHdomain is humanized.
 13. The polypeptide of claim 1, wherein at leastone VHH domain comprises an amino acid sequence at least 85%, at least90%, at least 95%, or at least 99% identical to the amino acid sequenceof SEQ ID NO: 38, 114, 39, 40, 41, 42, 43, 44, 45, 46, 115, 116, 117,118, 119, 120, 121, or
 122. 14. The polypeptide of claim 1, wherein atleast one VHH domain comprises the amino acid sequence of SEQ ID NO: 38,114, 39, 40, 41, 42, 43, 44, 45, 46, 115, 116, 117, 118, 119, 120, 121,or
 122. 15. The polypeptide of claim 1, wherein at least one VHH domaincomprises an amino acid sequence at least 85%, at least 90%, at least95%, at least 99% or 100% identical to the amino acid sequence of SEQ IDNO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 123, 124, 125, 126, 127, 128, 129,130,
 131. 16. (canceled)
 17. The polypeptide of claim 1, comprising one,two, or three VHH domains.
 18. (canceled)
 19. The polypeptide of claim1, wherein the polypeptide comprises at least one binding domain thatbinds an antigen other than CD33.
 20. The polypeptide of claim 19,wherein the polypeptide comprises at least one binding domain that bindsCD3, T-cell receptor (TCR) α, TCRβ, CD28, CD16, CD32A, CD64, CD89,NKp46, or NKG2D.
 21. The polypeptide of claim 17, wherein each VHHdomain binds CD33.
 22. (canceled)
 23. (canceled)
 24. (canceled)
 25. Thepolypeptide of claim 1, wherein the polypeptide comprises an Fc region.26. The polypeptide of claim 25, wherein the Fc region comprises anamino acid sequence selected from SEQ ID NOs: 74-109.
 27. Thepolypeptide of claim 25, which forms a dimer under physiologicalconditions.
 28. (canceled)
 29. (canceled)
 30. An immunoconjugatecomprising the polypeptide of claim 1 and a cytotoxic agent.
 31. Theimmunoconjugate of claim 30, wherein the cytotoxic agent is selectedfrom a calicheamicin, an auristatin, a dolastatin, a tubulicin, amaytansinoid, a cryptophycin, a duocarmycin, an esperamicin, apyrrolobenzodiazepine, and an enediyne antibiotic.
 32. A pharmaceuticalcomposition comprising the polypeptide of claim 1, and apharmaceutically acceptable carrier.
 33. An isolated nucleic acid thatencodes the polypeptide of claim
 1. 34. A vector comprising the nucleicacid of claim
 33. 35. (canceled)
 36. A host cell that expresses thepolypeptide of claim
 1. 37. A method of producing a polypeptide,comprising incubating the host cell of claim 36 under conditionssuitable for expression of the polypeptide, and isolating thepolypeptide.
 38. (canceled)
 39. A method of treating cancer comprisingadministering to a subject with cancer a pharmaceutically effectiveamount of the polypeptide of claim
 1. 40. The method of claim 39,wherein the cancer is selected from lymphoma; Hodgkin's lymphoma;non-Hodgkin's lymphoma; B-cell lymphoma; low grade/follicularnon-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; Waldenstrom's macroglobulinemia; chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acutemyeloid leukemia (AML); Hairy cell leukemia; and chronic myeloblasticleukemia.
 41. (canceled)
 42. The method of claim 36, further comprisingadministering an additional therapeutic agent.
 43. The method of claim42, wherein the additional therapeutic agent is an anti-cancer agent,wherein the anti-cancer agent is selected from a chemotherapeutic agent,an anti-cancer biologic, radiation therapy, CAR-T therapy, and anoncolytic virus.
 44. (canceled)
 45. (canceled)